Identification of gene specific cis-regulatory elements during differentiation of mouse embryonic stem cells: An integrative approach using high-throughput datasets.

Gene expression governs cell fate, and is regulated via a complex interplay of transcription factors and molecules that change chromatin structure. Advances in sequencing-based assays have enabled investigation of these processes genome-wide, leading to large datasets that combine information on the dynamics of gene expression, transcription factor binding and chromatin structure as cells differentiate. While numerous studies focus on the effects of these features on broader gene regulation, less work has been done on the mechanisms of gene-specific transcriptional control. In this study, we have focussed on the latter by integrating gene expression data for the in vitro differentiation of murine ES cells to macrophages and cardiomyocytes, with dynamic data on chromatin structure, epigenetics and transcription factor binding. Combining a novel strategy to identify communities of related control elements with a penalized regression approach, we developed individual models to identify the potential control elements predictive of the expression of each gene. Our models were compared to an existing method and evaluated using the existing literature and new experimental data from embryonic stem cell differentiation reporter assays. Our method is able to identify transcriptional control elements in a gene specific manner that reflect known regulatory relationships and to generate useful hypotheses for further testing.

Cooperative binding of AP-1 and TEAD4 modulates the balance between vascular smooth muscle and hemogenic cell fate.

The transmission of extracellular signals into the nucleus involves inducible transcription factors, but how different signalling pathways act in a cell type-specific fashion is poorly understood. Here, we studied the regulatory role of the AP-1 transcription factor family in blood development using embryonic stem cell differentiation coupled with genome-wide transcription factor binding and gene expression analyses. AP-1 factors respond to MAP kinase signalling and comprise dimers of FOS, ATF and JUN proteins. To examine genes regulated by AP-1 and to examine how it interacts with other inducible transcription factors, we abrogated its global DNA-binding activity using a dominant-negative FOS peptide. We show that FOS and JUN bind to and activate a specific set of vascular genes and that AP-1 inhibition shifts the balance between smooth muscle and hematopoietic differentiation towards blood. Furthermore, AP-1 is required for de novo binding of TEAD4, a transcription factor connected to Hippo signalling. Our bottom-up approach demonstrates that AP-1- and TEAD4-associated cis-regulatory elements form hubs for multiple signalling-responsive transcription factors and define the cistrome that regulates vascular and hematopoietic development by extrinsic signals.

NFATc1 releases BCL6-dependent repression of CCR2 agonist expression in peritoneal macrophages from Saccharomyces cerevisiae infected mice.

The link between the extensive usage of calcineurin (CN) inhibitors cyclosporin A and tacrolimus (FK506) in transplantation medicine and the increasing rate of opportunistic infections within this segment of patients is alarming. Currently, how peritoneal infections are favored by these drugs, which impair the activity of several signaling pathways including the Ca(++) /CN/NFAT, Ca(++) /CN/cofilin, Ca(++) /CN/BAD, and NF-κB networks, is unknown. Here, we show that Saccharomyces cerevisiae infection of peritoneal resident macrophages triggers the transient nuclear translocation of NFATc1ß isoforms, resulting in a coordinated, CN-dependent induction of the Ccl2, Ccl7, and Ccl12 genes, all encoding CCR2 agonists. CN inhibitors block the CCR2-dependent recruitment of inflammatory monocytes (IM) to the peritoneal cavities of S. cerevisiae infected mice. In myeloid cells, NFATc1/ß proteins represent the most prominent NFATc1 isoforms. NFATc1/ß ablation leads to a decrease of CCR2 chemokines, impaired mobilization of IMs, and delayed clearance of infection. We show that, upon binding to a composite NFAT/BCL6 regulatory element within the Ccl2 promoter, NFATc1/ß proteins release the BCL6-dependent repression of Ccl2 gene in macrophages. These findings suggest a novel CN-dependent cross-talk between NFAT and BCL6 transcription factors, which may affect the outcome of opportunistic fungal infections in immunocompromised patients.

RUNX1 reshapes the epigenetic landscape at the onset of haematopoiesis.

Cell fate decisions during haematopoiesis are governed by lineage-specific transcription factors, such as RUNX1, SCL/TAL1, FLI1 and C/EBP family members. To gain insight into how these transcription factors regulate the activation of haematopoietic genes during embryonic development, we measured the genome-wide dynamics of transcription factor assembly on their target genes during the RUNX1-dependent transition from haemogenic endothelium (HE) to haematopoietic progenitors. Using a Runx1-/- embryonic stem cell differentiation model expressing an inducible Runx1 gene, we show that in the absence of RUNX1, haematopoietic genes bind SCL/TAL1, FLI1 and C/EBPß and that this early priming is required for correct temporal expression of the myeloid master regulator PU.1 and its downstream targets. After induction, RUNX1 binds to numerous de novo sites, initiating a local increase in histone acetylation and rapid global alterations in the binding patterns of SCL/TAL1 and FLI1. The acquisition of haematopoietic fate controlled by Runx1 therefore does not represent the establishment of a new regulatory layer on top of a pre-existing HE program but instead entails global reorganization of lineage-specific transcription factor assemblies.

Dynamic epigenetic enhancer signatures reveal key transcription factors associated with monocytic differentiation states.

Cellular differentiation is orchestrated by lineage-specific transcription factors and associated with cell type-specific epigenetic signatures. In the present study, we used stage-specific, epigenetic "fingerprints" to deduce key transcriptional regulators of the human monocytic differentiation process. We globally mapped the distribution of epigenetic enhancer marks (histone H3 lysine 4 monomethylation, histone H3 lysine 27 acetylation, and the histone variant H2AZ), describe general properties of marked regions, and show that cell type-specific epigenetic "fingerprints" are correlated with specific, de novo-derived motif signatures at all of the differentiation stages studied (ie, hematopoietic stem cells, monocytes, and macrophages). We validated the novel, de novo-derived, macrophage-specific enhancer signature, which included ETS, CEBP, bZIP, EGR, E-Box and NF-κB motifs, by ChIP sequencing for a subset of motif corresponding transcription factors (PU.1, C/EBPß, and EGR2), confirming their association with differentiation-associated epigenetic changes. We describe herein the dynamic enhancer landscape of human macrophage differentiation, highlight the power of genome-wide epigenetic profiling studies to reveal novel functional insights, and provide a unique resource for macrophage biologists.

GFI1 and GFI1B control the loss of endothelial identity of hemogenic endothelium during hematopoietic commitment.

Recent studies have established that during embryonic development, hematopoietic progenitors and stem cells are generated from hemogenic endothelium precursors through a process termed endothelial to hematopoietic transition (EHT). The transcription factor RUNX1 is essential for this process, but its main downstream effectors remain largely unknown. Here, we report the identification of Gfi1 and Gfi1b as direct targets of RUNX1 and critical regulators of EHT. GFI1 and GFI1B are able to trigger, in the absence of RUNX1, the down-regulation of endothelial markers and the formation of round cells, a morphologic change characteristic of EHT. Conversely, blood progenitors in Gfi1- and Gfi1b-deficient embryos maintain the expression of endothelial genes. Moreover, those cells are not released from the yolk sac and disseminated into embryonic tissues. Taken together, our findings demonstrate a critical and specific role of the GFI1 transcription factors in the first steps of the process leading to the generation of hematopoietic progenitors from hemogenic endothelium.

Early chromatin unfolding by RUNX1: a molecular explanation for differential requirements during specification versus maintenance of the hematopoietic gene expression program.

At the cellular level, development progresses through successive regulatory states, each characterized by their specific gene expression profile. However, the molecular mechanisms regulating first the priming and then maintenance of gene expression within one developmental pathway are essentially unknown. The hematopoietic system represents a powerful experimental model to address these questions and here we have focused on a regulatory circuit playing a central role in myelopoiesis: the transcription factor PU.1, its target gene colony-stimulating-factor 1 receptor (Csf1r), and key upstream regulators such as RUNX1. We find that during ontogeny, chromatin unfolding precedes the establishment of active histone marks and the formation of stable transcription factor complexes at the Pu.1 locus and we show that chromatin remodeling is mediated by the transient binding of RUNX1 to Pu.1 cis-elements. By contrast, chromatin reorganization of Csf1r requires prior expression of PU.1 together with RUNX1 binding. Once the full hematopoietic program is established, stable transcription factor complexes and active chromatin can be maintained without RUNX1. Our experiments therefore demonstrate how individual transcription factors function in a differentiation stage-specific manner to differentially affect the initiation versus maintenance of a developmental program.

Chromatin regulation by RUNX1.

The transcription factor RUNX1 is essential for definitive hematopoiesis and is required for the expression of a number of important hematopoietic regulator genes. It was recently shown that RUNX1 acts within a narrow developmental window during which it cannot be replaced by other members of the RUNX transcription factor family. Studies of the molecular basis of this phenomenon revealed that RUNX1 is required for the opening of chromatin of important hematopoietic regulator genes and for the formation, but not the maintenance of stable transcription factor complexes on these genes. However, the chromatin opening activity of RUNX1 is context dependent, indicating that it cooperates with alternate transcription factors at different stages of hematopoietic development. This review summarizes recent results on the regulation of chromatin structure by RUNX1 in developing hematopoietic cells.

Transcriptomic profiling identifies a PU.1 regulatory network in macrophages.

PU.1 is a key transcription factor for hematopoiesis and macrophage differentiation. Using chromatin immunoprecipitation we have previously identified several PU.1 target genes in macrophages and microglia. With the aim to complement these studies, we performed a transcriptomic analysis of PU.1(-/-) progenitors after restoration of PU.1 activity. PUER cells committed to macrophage differentiation were analyzed with novel Affymetrix exon 1.0 ST arrays and Affymetrix 430 2.0 genome arrays for crosswise validation. We combined these genome-wide expression data with a publicly-available microarray dataset of PU.1-knockdown hematopoietic stem cells for an integrated analysis. Bibliographic gene connections, binding site prediction and ChIP-Chip data were used to define a multi-level PU.1 regulatory network in macrophages. Moreover, an alternative transcript of the novel PU.1 target gene Ptpro was identified by exon arrays and PU.1 binding to an intronic promoter was demonstrated. In conclusion, we present a PU.1 transcriptional network with novel validated PU.1 target genes.

PU.1 and ICSBP control constitutive and IFN-gamma-regulated Tlr9 gene expression in mouse macrophages.

Macrophages are activated by unmethylated CpG-containing DNA (CpG DNA) via TLR9. IFN-gamma and LPS can synergize with CpG DNA to enhance proinflammatory responses in murine macrophages. Here, we show that LPS and IFN-gamma up-regulated Tlr9 mRNA in murine bone marrow-derived macrophages (BMM). The ability of LPS and IFN-gamma to induce Tlr9 mRNA expression in BMM was dependent on the presence of the growth factor, CSF-1, which is constitutively present in vivo. However, there were clear differences in mechanisms of Tlr9 mRNA induction. LPS stimulation rapidly removed the CSF-1 receptor (CSF-1R) from the cell surface, thereby blocking CSF-1-mediated transcriptional repression and indirectly inducing Tlr9 mRNA expression. By contrast, IFN-gamma activated the Tlr9 promoter directly and only marginally affected cell surface CSF-1R expression. An approximately 100-bp proximal promoter of the murine Tlr9 gene was sufficient to confer basal and IFN-gamma-inducible expression in RAW264.7 cells. A composite IFN regulatory factor (IRF)/PU.1 site upon the major transcription start site was identified. Mutation of the binding sites for PU.1 or IRF impaired basal promoter activity, but only the IRF-binding site was required for IFN-gamma induction. The mRNA expression of the IRF family member IFN consensus-binding protein [(ICSBP)/IRF8] was coregulated with Tlr9 in macrophages, and constitutive and IFN-gamma-inducible Tlr9 mRNA expression was reduced in ICSBP-deficient BMM. This study therefore characterizes the regulation of mouse Tlr9 expression and defines a molecular mechanism by which IFN-gamma amplifies mouse macrophage responses to CpG DNA.

The Co-operation of RUNX1 with LDB1, CDK9 and BRD4 Drives Transcription Factor Complex Relocation During Haematopoietic Specification.

Haematopoietic cells arise from endothelial cells within the dorsal aorta of the embryo via a process called the endothelial-haematopoietic transition (EHT). This process crucially depends on the transcription factor RUNX1 which rapidly activates the expression of genes essential for haematopoietic development. Using an inducible version of RUNX1 in a mouse embryonic stem cell differentiation model we showed that prior to the EHT, haematopoietic genes are primed by the binding of the transcription factor FLI1. Once expressed, RUNX1 relocates FLI1 towards its binding sites. However, the nature of the transcription factor assemblies recruited by RUNX1 to reshape the chromatin landscape and initiate mRNA synthesis are unclear. Here, we performed genome-wide analyses of RUNX1-dependent binding of factors associated with transcription elongation to address this question. We demonstrate that RUNX1 induction moves FLI1 from distal ETS/GATA sites to RUNX1/ETS sites and recruits the basal transcription factors CDK9, BRD4, the Mediator complex and the looping factor LDB1. Our study explains how the expression of a single transcription factor can drive rapid and replication independent transitions in cellular shape which are widely observed in development and disease.

Dynamic Gene Regulatory Networks Drive Hematopoietic Specification and Differentiation.

Metazoan development involves the successive activation and silencing of specific gene expression programs and is driven by tissue-specific transcription factors programming the chromatin landscape. To understand how this process executes an entire developmental pathway, we generated global gene expression, chromatin accessibility, histone modification, and transcription factor binding data from purified embryonic stem cell-derived cells representing six sequential stages of hematopoietic specification and differentiation. Our data reveal the nature of regulatory elements driving differential gene expression and inform how transcription factor binding impacts on promoter activity. We present a dynamic core regulatory network model for hematopoietic specification and demonstrate its utility for the design of reprogramming experiments. Functional studies motivated by our genome-wide data uncovered a stage-specific role for TEAD/YAP factors in mammalian hematopoietic specification. Our study presents a powerful resource for studying hematopoiesis and demonstrates how such data advance our understanding of mammalian development.

Activation of the signal transducer and activator of transcription 1 signaling pathway in thymocytes from HIV-1-infected human thymus.

OBJECTIVE: To identify HIV-induced host factors in the severe combined immunodeficient (SCID)-hu Thy/Liv mouse that may contribute to HIV pathogenesis in the thymus. DESIGN: To identify genes specifically altered by HIV-1 infection using the cDNA microarray assay, SCID-hu Thy/Liv organs derived from the same donors were used. Therefore, no genetic variations existed between HIV and mock-infected samples. In addition, the 12-14 day post-infection timepoint was chosen because no significant thymocyte depletion was detected in HIV-infected Thy/Liv organs, so mRNA from the same cell types could be compared. METHODS: Using SCID-hu Thy/Liv mice constructed from the same donor tissues, we analysed the expression of 9183 host genes in response to HIV infection with cDNA microarrays. Expression of selected genes with more than threefold induction was confirmed by measuring RNA (reverse transcriptase-polymerase chain reaction; RT-PCR) and proteins. RESULTS: HIV-1 (JD or NL4-3) infection of the SCID-hu Thy/Liv mouse led to more than threefold induction of 19 genes, 12 of which were IFN-inducible and six were unknown EST clones. We confirmed induction by RT-PCR and protein blots. Both signal transducer and activator of transcription (STAT)1 and STAT2 proteins were induced, and STAT1 was also activated by phosphorylation at the Tyr701 and Ser727 sites in human thymus infected with HIV-JD or NL4-3. Treatment of human fetal thymus organ culture or human thymocytes with recombinant HIV-1 gp120 proteins also led to induction or activation of STAT1. CONCLUSION: HIV-1 infection of the thymus led to activation of the STAT1 signaling pathway in thymocytes, which may contribute to HIV-1 pathogenesis in the thymus.

Transcriptional effects of colony-stimulating factor-1 in mouse macrophages.

Colony-stimulating factor-1 (CSF-1) is a major regulator of macrophage development. CSF-1-dependent signalling has been implicated in proliferation, survival, and differentiation of mononuclear phagocytes, however, relatively little is known about the effects of CSF-1 on macrophage gene transcription and on CSF-1-responsive gene promoters. We used a combination of transcription profiling and in silico motif search to characterize genes that are regulated in mature bone marrow-derived macrophages cultured in the presence or absence of CSF-1. The expression of many known differentiation-associated macrophage markers was not significantly affected in the absence of CSF-1. Genes repressed by CSF-1 comprised a considerable number of granulocyte-specific genes. The respective gene promoters; however, were not significantly enriched for specific DNA patterns, suggesting that these genes are regulated by promoter-distal elements or at a post-transcriptional level. Genes downregulated upon CSF-1 deprivation showed a highly significant association with cell division which is in line with the known role of CSF-1 as a proliferation stimulus for mouse macrophages. Interestingly, three DNA patterns were significantly co-enriched in CSF-1-dependent gene promoters, including motifs related to NFY, CHR, and E2F sites. These motifs showed a strong positional preference on target promoters at -60, -30 and 0 bp upstream of the transcription start site, and define the common promoter structure of CSF-1-responsive genes.

Transcription factor PU.1 controls transcription start site positioning and alternative TLR4 promoter usage.

Human and mouse show markedly different sensitivities toward bacterial endotoxins, and recent evidence suggests that a species-specific regulation of the lipopolysaccharide receptor Toll-like receptor 4 (Tlr4) may contribute to this phenomenon. To gain further insight into mechanisms of Tlr4 regulation, we conducted a detailed in vivo and in vitro study of the murine Tlr4 gene, including analysis of transcription start site location, transcription factor occupancy, and activities of its proximal regulatory sequences. Our analyses identified a PU.1-dependent myeloid promoter, which is conserved between humans and mouse. We also identified an additional, distal promoter, located approximately 200 bp upstream of the myeloid-specific promoter, which is a functional target of E-box binding factors. In contrast to humans, where non-myeloid cells utilize both promoters, the distal Tlr4 promoter initiates all Tlr4 transcripts in murine non-myeloid cells, indicating that species-specific differences in TLR4 mRNA regulation may primarily exist in non-myeloid cell types. Interestingly, PU.1 null murine myeloid progenitor cells predominantly use the distal promoter, and the conditional induction of PU.1 expression in these cells leads to the rapid switch of transcription initiation to the proximal myeloid promoter. This indicates a direct role for PU.1 in determining the transcriptional start site and in recruiting the basal transcription machinery to myeloid promoters.

CCAAT enhancer-binding protein beta regulates constitutive gene expression during late stages of monocyte to macrophage differentiation.

Human monocyte to macrophage differentiation is accompanied by pronounced phenotypical changes and generally proceeds in the absence of proliferation. The molecular events governing this process are poorly understood. Here, we studied the regulation of the macrophage-specific chitotriosidase (CHIT1) gene promoter to gain insights into the mechanisms of transcriptional control during the differentiation of human blood monocytes into macrophages. We used transient transfections to define a cell type-specific minimal promoter that was mainly dependent on a proximal C/EBP motif that bound multiple C/EBP factors in gel shift assays. In depth analysis of occupied promoter elements using in vivo footprinting and chromatin immunoprecipitation analyses demonstrated the differentiation-associated recruitment of C/EBPbeta and PU.1 at the proximal promoter in parallel with CHIT1 mRNA induction. Notably, the induction of C/EBPbeta promoter binding strongly correlated with increased nuclear levels of Thr-235-phosphorylated C/EBPbeta protein during the differentiation process, whereas C/EBPbeta mRNA and total protein expression remained relatively stable. Our data suggest an important constitutive gene regulatory function for C/EBPbeta in differentiated macrophages but not in human blood monocytes.