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Prostaglandin E2 (PGE 2 ) and 16,16-dimethyl-PGE 2 (dmPGE 2 ) are important regulators of hematopoietic stem and progenitor cell (HSPC) fate and offer potential to enhance stem cell therapies [C. Cutler et al. Blood 122 , 3074–3081(2013); W. Goessling et al. Cell Stem Cell 8 , 445–458 (2011); W. Goessling et al. Cell 136 , 1136–1147 (2009)]. Here, we report that PGE 2 -induced changes in chromatin at enhancer regions through histone-variant H2A.Z permit acute inflammatory gene induction to promote HSPC fate. We found that dmPGE 2 -inducible enhancers retain MNase-accessible, H2A.Z-variant nucleosomes permissive of CREB transcription factor (TF) binding. CREB binding to enhancer nucleosomes following dmPGE 2 stimulation is concomitant with deposition of histone acetyltransferases p300 and Tip60 on chromatin. Subsequent H2A.Z acetylation improves chromatin accessibility at stimuli-responsive enhancers. Our findings support a model where histone-variant nucleosomes retained within inducible enhancers facilitate TF binding. Histone-variant acetylation by TF-associated nucleosome remodelers creates the accessible nucleosome landscape required for immediate enhancer activation and gene induction. Our work provides a mechanism through which inflammatory mediators, such as dmPGE 2 , lead to acute transcriptional changes and modify HSPC behavior to improve stem cell transplantation.

Prostaglandin E2 (PGE2) and 16,16-dimethyl-PGE2 (dmPGE2) are important regulators of hematopoietic stem and progenitor cell (HSPC) fate and offer potential to enhance stem cell therapies1,2. The mechanism of gene regulation in response to dmPGE2 is poorly understood. Here, we show that dmPGE2 regulates inflammatory gene induction by modulating the chromatin architecture and activity of enhancer elements in human HSPCs. We identified the specific genomic reorganization at stimuli-responsive enhancers that permits rapid transcriptional activation. We found that dmPGE2-inducible enhancers retain MNase-accessible, H2A.Z-variant nucleosomes that are permissive to binding of the transcription factor CREB. CREB binding to enhancer nucleosomes is concomitant with deposition of the histone acetyltransferases p300 and Tip60 on chromatin. Subsequent H2A.Z acetylation improves chromatin accessibility at stimuli-responsive enhancers. Our findings support a model where histone variant nucleosomes retained within inducible enhancers facilitate transcription factor (TF) binding. Acetylation of histone variant nucleosomes by TF-associated nucleosome remodelers creates the accessible nucleosome landscape required for immediate enhancer activation and gene induction. Our work provides a mechanism by which inflammatory mediators such as dmPGE2 lead to acute transcriptional changes and alter HSPC behavior.

Empirical and anecdotal evidence has associated stress with accelerated hair greying (formation of unpigmented hairs)1,2, but so far there has been little scientific validation of this link. Here we report that, in mice, acute stress leads to hair greying through the fast depletion of melanocyte stem cells. Using a combination of adrenalectomy, denervation, chemogenetics3,4, cell ablation and knockout of the adrenergic receptor specifically in melanocyte stem cells, we find that the stress-induced loss of melanocyte stem cells is independent of immune attack or adrenal stress hormones. Instead, hair greying results from activation of the sympathetic nerves that innervate the melanocyte stem-cell niche. Under conditions of stress, the activation of these sympathetic nerves leads to burst release of the neurotransmitter noradrenaline (also known as norepinephrine). This causes quiescent melanocyte stem cells to proliferate rapidly, and is followed by their differentiation, migration and permanent depletion from the niche. Transient suppression of the proliferation of melanocyte stem cells prevents stress-induced hair greying. Our study demonstrates that neuronal activity that is induced by acute stress can drive a rapid and permanent loss of somatic stem cells, and illustrates an example in which the maintenance of somatic stem cells is directly influenced by the overall physiological state of the organism. Stress induces hair greying in mice through depletion of melanocyte stem cells, which is mediated by the activation of sympathetic nerves rather than through immune attack or adrenal stress hormones.

Hematopoietic stem cells (HSCs) must ensure adequate blood cell production following distinct external stressors. A comprehensive understanding of in vivo heterogeneity and specificity of HSC responses to external stimuli is currently lacking. We performed single-cell RNA sequencing (scRNA-Seq) on functionally validated mouse HSCs and LSK (Lin-, c-Kit+, Sca1+) progenitors after in vivo perturbation of niche signals interferon, granulocyte-colony stimulating factor (G-CSF), and prostaglandin. We identified six HSC states that are characterized by enrichment but not exclusive expression of marker genes. Niche perturbations induce novel and rapid transitions between these HSC states. Differential expression analysis within each state revealed HSC- and LSK-specific molecular signatures for each perturbation. Chromatin analysis of unperturbed HSCs and LSKs by scATAC-Seq revealed HSC-specific, cell intrinsic predispositions to niche signals. We compiled a comprehensive resource of HSC- and progenitor-specific chromatin and transcriptional features that represent important determinants of regenerative potential during stress hematopoiesis.

Epoxyeicosatrienoic acids (EETs) are lipid-derived signaling molecules with cardioprotective and vasodilatory actions. We recently showed that 11,12-EET enhances hematopoietic induction and engraftment in mice and zebrafish. EETs are known to signal via G protein-coupled receptors, with evidence supporting the existence of a specific high-affinity receptor. Identification of a hematopoietic-specific EET receptor would enable genetic interrogation of EET signaling pathways, and perhaps clinical use of this molecule. We developed a bioinformatic approach to identify an EET receptor based on the expression of G protein-coupled receptors in cell lines with differential responses to EETs. We found 10 candidate EET receptors that are expressed in three EET-responsive cell lines, but not expressed in an EET-unresponsive line. Of these, only recombinant GPR132 showed EET-responsiveness in vitro, using a luminescence-based β-arrestin recruitment assay. Knockdown of zebrafish gpr132b prevented EET-induced hematopoiesis, and marrow from GPR132 knockout mice showed decreased long-term engraftment capability. In contrast to high-affinity EET receptors, GPR132 is reported to respond to additional hydroxy-fatty acids in vitro, and we found that these same hydroxy-fatty acids enhance hematopoiesis in the zebrafish. We conducted structure–activity relationship analyses using both cell culture and zebrafish assays on diverse medium-chain fatty acids. Certain oxygenated, unsaturated free fatty acids showed high activation of GPR132, whereas unoxygenated or saturated fatty acids had lower activity. Absence of the carbon-1 position carboxylic acid prevented activity, suggesting that this moiety is required for receptor activation. GPR132 responds to a select panel of oxygenated polyunsaturated fatty acids to enhance both embryonic and adult hematopoiesis.

This perspective describes the usefulness of zebrafish as a model to study interaction of hematopoietic stem cells with the associated niche in vivo, explains how such interactions influence regeneration, migration, and clonality of HSCs, and defines their fate during differentiation.

Wolbachia are intracellular bacteria that infect invertebrates at pandemic levels, including insect vectors of devastating infectious diseases. Although Wolbachia are providing novel strategies for the control of several human pathogens, the processes underlying Wolbachia ’s successful propagation within and across species remain elusive. Wolbachia are mainly vertically transmitted; however, there is also evidence of extensive horizontal transmission. Here, we provide several lines of evidence supporting Wolbachia ’s targeting of ovarian stem cell niches—referred to as “niche tropism”—as a previously overlooked strategy for Wolbachia thriving in nature. Niche tropism is pervasive in Wolbachia infecting the Drosophila genus, and different patterns of niche tropism are evolutionarily conserved. Phylogenetic analysis, confirmed by hybrid introgression and transinfection experiments, demonstrates that bacterial factors are the major determinants of differential patterns of niche tropism. Furthermore, bacterial load is increased in germ-line cells passing through infected niches, supporting previous suggestions of a contribution of Wolbachia from stem-cell niches toward vertical transmission. These results support the role of stem-cell niches as a key component for the spreading of Wolbachia in the Drosophila genus and provide mechanistic insights into this unique tissue tropism.

© 2016 by The American Society of Hematology. Enhancers are the primary determinants of cell identity, and specific promoter/enhancer combinations of Endoglin (ENG) have been shown to target blood and endothelium in the embryo. Here, we generated a series of embryonic stem cell lines, each targeted with reporter constructs driven by specific promoter/enhancer combinations of ENG, to evaluate their discriminative potential and value as molecular probes of the corresponding transcriptome. The Eng promoter (P) in combination with the 28/17/19-kb enhancers, targeted cells in FLK1 mesoderm that were enriched for blast colony forming potential, whereas the P/28-kb enhancer targeted TIE21/c-KIT1/CD412 endothelial cells that were enriched for hematopoieticpotential. These fractions were isolated using reporter expression and their transcriptomes profiled by RNA-seq. There was high concordance between our signatures and those from embryos with defects at corresponding stages of hematopoiesis. Of the 6 genes that were upregulated in both hemogenic mesoderm and hemogenic endothelial fractions targeted by the reporters,LRP2, amultiligandreceptor,wasthe only gene that hadnot previously been associated with hematopoiesis.WeshowthatLRP2is indeedinvolved indefinitivehematopoiesisandbydoingsovalidatetheuseof reportergene-coupled enhancers as probes to gain insights into transcriptional changes that facilitate cell fate transitions.

We tested the use of the small-molecule Inhibitor of Apoptosis Protein (IAP) inhibitor LBW242 in combination with the standard-of-care therapies of irradiation and temozolomide for malignant gliomas. In vitro assays demonstrated that LBW242 enhanced the cytotoxic activity of radiotherapy, and clonogenic assays showed that the combination therapy led to a synergistic anti-glioma effect in multiple cell lines. Neurosphere assays revealed that the combination of radiation and LBW242 led to a pro-apoptotic effect in these glioma–initiating cell-enriched assays, with a corresponding inhibition of primary tumor cell growth. Athymic mice bearing established human malignant glioma tumor xenografts treated with LBW242 plus radiation and temozolomide demonstrated a synergistic suppression of tumor growth. Taken together, these experiments show that the pro-apoptotic and anti-glioma effects of radiotherapy and chemotherapy can be enhanced by the addition of a small-molecule IAP inhibitor. These results are readily translatable to clinical trial and offer the potential for improved treatment outcomes for patients with glioma.

Malignant gliomas are highly lethal tumors resistant to current therapies. The standard treatment modality for these tumors, surgical resection followed by radiation therapy and concurrent temozolomide, has demonstrated activity, but development of resistance and disease progression is common. Although oncogenic Ras mutations are uncommon in gliomas, Ras has been found to be constitutively activated through the action of upstream signaling pathways, suggesting that farnesyltransferase inhibitors may show activity against these tumors. We now report the in vitro and orthotopic in vivo results of combination therapy using radiation, temozolomide and lonafarnib (SCH66336), an oral farnesyl transferase inhibitor, in a murine model of glioblastoma. We examined the viability, proliferation, farnesylation of H-Ras, and activation of downstream signaling of combination-treated U87 cells in vitro. Lonafarnib alone or in combination with radiation and temozolomide had limited tumor cell cytotoxicity in vitro although it did demonstrate significant inhibition in tumor cell proliferation. In vivo, lonafarnib alone had a modest ability to inhibit orthotopic U87 tumors, radiation and temozolomide demonstrated better inhibition, while significant anti-tumor activity was found with concurrent lonafarnib, radiation, and temozolomide, with the majority of animals demonstrating a decrease in tumor volume. The use of tumor neurospheres derived from freshly resected adult human glioblastoma tissue was relatively resistant to both temozolomide and radiation therapy. Lonafarnib had a significant inhibitory activity against these neurospheres and could potentate the activity of temozolomide and radiation. These data support the continued research of high grade glioma treatment combinations of farnesyl transferase inhibitors, temozolomide, and radiation therapy.

Stimulation of hematopoietic stem and progenitor cells (HSPCs) with the inflammatory mediator Prostaglandin E2 (PGE2) enhances self-renewal and stem cell engraftment following transplantation. Currently, the long-acting derivative of PGE2, 16,16-dimethyl-PGE2 (dmPGE2) is in its fourth clinical trial to improve HSC engraftment and reduce graft versus host disease. To understand the effect of dmPGE2, we assessed genome-wide chromatin reorganization and gene expression changes in human CD34+ HSPCs after 2 hours of dmPGE2 treatment, the time period of treatment in the clinical trials. Enhancers are known to regulate gene expression changes in specific environmental contexts such as stress or inflammation, however the regulatory principles by which subsets of enhancers become activated are poorly understood. Here, we mapped active enhancers by ChIP-seq for H2K27ac and found that dmPGE2 activates a discrete set of enhancers in HSPCs. To investigate enhancer chromatin remodeling, we performed micrococcal nuclease digestion followed by high-throughput sequencing (MNase-seq) to map the occupancy and position of nucleosomes. We found that, contrary to the predominant assumption that open chromatin structures are essentially nucleosome-free, MNase-accessible nucleosomes are retained at inducible enhancers following dmPGE2 stimulation. Through ATAC-seq analysis we mapped changes in open chromatin and found that induced enhancers gain chromatin accessibility following stimulation while maintaining their nucleosome configuration. Surprisingly, this indicates that nucleosomes present at the center of dmPGE2-responsive enhancers play an important function in enhancer accessibility and activity. We then correlated enhancers with gene expression changes by performing RNA-seq and found that genes associated with dmPGE2-induced enhancers display higher gene expression changes after stimulation compared to genes associated with non-responsive enhancers. Transcripts upregulated after dmPGE2 treatment include previously identified regulators of self-renewal and migration such as NR4A2, EGR1 and CXCR4. Moreover, inflammatory chemokines including CXCL2 and CXCL8 as well as members of the activating protein 1 (AP-1) transcription factor gene family such as FOS, FOSL2 and JUNB are increasingly expressed upon stimulation. The gene expression profile included a signature implying CREB as the main transcription factor responsible for the acute dmPGE2 response. Western blot revealed dmPGE2-mediated activation of the signaling transcription factor CREB through phosphorylation in HSPCs. Using ChIP-seq, we found increased genomic binding of phospho-CREB (pCREB) after dmPGE2 treatment in the enhancers. Surprisingly, the binding of pCREB coincided directly with variant histone H2A.Z containing labile nucleosomes in enhancers. We validated the interaction between pCREB and H2A.Z on chromatin in dmPGE2-responsive U937 cells through chromatin fractionation followed by complex immunoprecipitation. This suggests that labile nucleosomes provide sufficient DNA access to allow for binding of pCREB at enhancers. Taken together, our study proposes a novel model for stimulus-mediated activation of enhancers by the inflammatory mediator dmPGE2. dmPGE2 induces the phosphorylation of CREB and subsequently leads to a specific interaction of pCREB with previously deposited H2A.Z-rich nucleosomes at inducible enhancers who regulate genes that promote HSPC fate. This new mechanism of variant histone deposition followed by the interaction with a signaling transcription factor at enhancers supports a rapid inducible response from the environment. Disclosures No relevant conflicts of interest to declare.

Forward genetic screens in genetically accessible invertebrate organisms such asDrosophila melanogasterhave shed light on transcription factors that specify formation of neurons in the vertebrate CNS. However, invertebrate models have, to date, been uninformative with respect to genes that specify formation of the vertebrate glial lineages. All recent insights into specification of vertebrate glia have come via monitoring the spatial and temporal expression patterns of individual transcription factors during development. In studies described here, we have taken this approach to the genome scale with anin silicoscreen of the Mahoney pictorial atlas of transcription factor expression in the developing CNS. From the population of 1445 known or probable transcription factors encoded in the mouse genome, we identify 12 novel transcription factors that are expressed in glial lineage progenitor cells. Entry-level screens for biological function establish one of these transcription factors, Klf15, as sufficient for genesis of precocious GFAP-positive astrocytes in spinal cord explants. Another transcription factor, Tcf4, plays an important role in maturation of oligodendrocyte progenitors.

The ability of cells to count and remember their divisions could underlie many alterations that occur during development, aging, and disease. We tracked the cumulative divisional history of slow-cycling hematopoietic stem cells (HSCs) throughout adult life. This revealed a fraction of rarely dividing HSCs that contained all the long-term HSC (LT-HSC) activity within the aging HSC compartment. During adult life, this population asynchronously completes four traceable symmetric self-renewal divisions to expand its size before entering a state of dormancy. We show that the mechanism of expansion involves progressively lengthening periods between cell divisions, with long-term regenerative potential lost upon a fifth division. Our data also show that age-related phenotypic changes within the HSC compartment are divisional history dependent. These results suggest that HSCs accumulate discrete memory stages over their divisional history and provide evidence for the role of cellular memory in HSC aging.

Throughout the lifetime of an individual, hematopoietic stem cells (HSCs) self-renew and differentiate into lineages that include erythrocytes, platelets and all immune cells. HSC transplantation offers a potentially curative treatment for a number of hematopoietic and non-hematopoietic malignancies as well as immune and genetic disorders. Limited availability of immune-matched donors reduces the viable options for many patients in need of HSC transplantation, particularly those of diverse racial and ethnic backgrounds. Due to rapid availability and less stringent immune-matching requirements, umbilical cord blood (UCB) has emerged as a valuable source of transplantable HSCs. A single UCB unit contains a suboptimal number of HSCs for treating larger children or adults and there has thus been great clinical interest in expanding UCB HSCs ex vivo for use in transplantation. In this review we discuss the latest research and future avenues for the therapeutic use of small lipid mediator dmPGE2 to expand HSC numbers for transplantation. Originally identified in a chemical screen in zebrafish, dmPGE2 has now advanced to a phase II clinical trial as a therapy for patients with leukemia and lymphoma who are undergoing UCB transplantation.

Analysis of gene expression in thousands of single cells generates a model of the blood regulatory network.

Wolbachia are widespread maternally-transmitted intracellular bacteria that infect most insect species and are able to alter the reproduction of innumerous hosts. The cellular bases of these alterations remain largely unknown. Here we report that Drosophila mauritiana infected with a native Wolbachia wMau strain produces about four times more eggs than the non-infected counterpart. Wolbachia infection leads to an increase in the mitotic activity of germline stem cells (GSCs) as well as a decrease in programmed cell death in the germarium. Our results suggest that upregulation of GSCs division is mediated by a tropism of Wolbachia for the germline stem cell niche (GSCN), the cellular microenvironment that supports GSCs.

Hematopoietic stem cells (HSCs) offer promising treatment options for many blood diseases. We have previously identified Prostaglandin E2 (PGE2), a small molecule that increased HSC numbers in the zebrafish embryo. In an adult mammalian transplantation setting a two hour treatment significantly enhanced HSC engraftment. Currently PGE2 is being tested in a phase 2 clinical trial to improve cord blood transplants. To better understand PGE2 effect on HSCs mouse multipotent progenitors (MPP), short term (ST) HSCs, and long term (LT) HSCs were isolated via FACS and given a two hour pulse of PGE2 followed by a competitive transplantation assay. Surprisingly, PGE2 treatment mainly affected ST-HSCs by dramatically prolonging their ability to contribute to peripheral blood. The effect of the two hour treatment persisted through secondary competitive transplants in which robust peripheral blood chimerism of ST-HSCs was evident even 1.5 years after having been exposed to the drug. To elucidate underlying molecular changes gene expression right after PGE2 treatment as well as in ST-HSCs after transplantation was assessed. PGE2 target genes were divided into two categories; "transiently induced" and "permanently induced" genes. Most of the transcripts upregulated two hour after PGE2 treatment were "transiently induced" meaning that they did not continue to be differentially expressed after transplantation. In contrast, a few transcripts including chemokines such as Cxcl2, Cxcl3, members of the Fos gene family as well as Nr4a1, 2 and 3 were both upregulated right after PGE2 treatment as well as in ST-HSCs after transplantation. We classified these genes as "permanently induced". ATAC (Assay for Transposase-Accessible Chromatin)-seq analysis of the transplanted PGE2 treated cells indicated that these "permanently induced" genes maintained a distinctly open chromatin profile in both promotor and enhancer regions, whereas the "transiently induced" genes did not. Gene expression in human CD34+ cells included a signature implying CREB as the main transcription factor responsible for the acute PGE2 response. Phospho-FACS in mouse ST-HSCs and Western-blot analysis in human CD34+ cells confirmed a significant increase in CREB phosphorylation after PGE2 stimulation. Chromatin immunoprecipitation (ChIP)-seq analysis of pCREB was able to identify specific genomic regions where pCREB is recruited to after PGE2 treatment. Compared to unstimulated CD34+ cells an increased binding of pCREB could be detected in promotor regions near transcription start sites. In addition over 90% of de-novo pCREB binding occurred in intergenic and intronic regions. To determine the activation state of these putative enhancers changes in the histone mark H3K27ac and open chromatin state (via ATAC-seq) were assessed after PGE2 treatment. The data suggest that PGE2-induced pCREB binding correlates with remodeling of chromatin already after two hours of drug treatment. Furthermore chromatin sites opened by PGE2 were significantly enriched for the CREB motif both in human CD34+ cells acutely after treatment as well as in mouse ST-HSCs 1.5 years after transplant. In summary this work shows that a two hour treatment with PGE2 is sufficient to confer long-term engraftment properties to ST-HSCs. PGE triggers a chromatin remodeling event through CREB that can permanently alter epigenetic state and gene expression of ST-HSCs. Understanding the self-renewal network induced by PGE2 will not only enrich current clinical applications targeted at increasing engraftable HSC numbers but also further basic understanding of HSC self-renewal. Disclosures Zon: FATE Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Scholar Rock: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder.