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Methods for single-cell RNA sequencing (scRNA-seq) have greatly advanced in recent years. While droplet- and well-based methods have increased the capture frequency of cells for scRNA-seq, these technologies readily produce technical artifacts, such as doublet cell captures. Doublets occurring between distinct cell types can appear as hybrid scRNA-seq profiles, but do not have distinct transcriptomes from individual cell states. We introduce DoubletDecon, an approach that detects doublets with a combination of deconvolution analyses and the identification of unique cell-state gene expression. We demonstrate the ability of DoubletDecon to identify synthetic, mixed-species, genetic, and cell-hashing cell doublets from scRNA-seq datasets of varying cellular complexity with a high sensitivity relative to alternative approaches. Importantly, this algorithm prevents the prediction of valid mixed-lineage and transitional cell states as doublets by considering their unique gene expression. DoubletDecon has an easy-to-use graphical user interface and is compatible with diverse species and unsupervised population detection algorithms., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Aging is associated with functional decline of hematopoietic stem cells (HSC) as well as an increased risk of myeloid malignancies. We performed an integrative characterization of epigenomic and transcriptomic changes, including single-cell RNA sequencing, during normal human aging. Lineage - CD34 + CD38 - cells [HSC-enriched (HSCe)] undergo age-associated epigenetic reprogramming consisting of redistribution of DNA methylation and reductions in H3K27ac, H3K4me1, and H3K4me3. This reprogramming of aged HSCe globally targets developmental and cancer pathways that are comparably altered in acute myeloid leukemia (AML) of all ages, encompassing loss of 4,646 active enhancers, 3,091 bivalent promoters, and deregulation of several epigenetic modifiers and key hematopoietic transcription factors, such as KLF6, BCL6, and RUNX3. Notably, in vitro downregulation of KLF6 results in impaired differentiation, increased colony-forming potential, and changes in expression that recapitulate aging and leukemia signatures. Thus, age-associated epigenetic reprogramming may form a predisposing condition for the development of age-related AML. SIGNIFICANCE: AML, which is more frequent in the elderly, is characterized by epigenetic deregulation. We demonstrate that epigenetic reprogramming of human HSCs occurs with age, affecting cancer and developmental pathways. Downregulation of genes epigenetically altered with age leads to impairment in differentiation and partially recapitulates aging phenotypes. This article is highlighted in the In This Issue feature, p. 983 ., (©2019 American Association for Cancer Research.)

Disruption of epigenetic regulation is a hallmark of acute myeloid leukemia (AML), but epigenetic therapy is complicated by the complexity of the epigenome. Herein, we developed a long-term primary AML ex vivo platform to determine whether targeting different epigenetic layers with 5-azacytidine and LSD1 inhibitors would yield improved efficacy. This combination was most effective in TET2 mut AML, where it extinguished leukemia stem cells and particularly induced genes with both LSD1-bound enhancers and cytosine-methylated promoters. Functional studies indicated that derepression of genes such as GATA2 contributes to drug efficacy. Mechanistically, combination therapy increased enhancer-promoter looping and chromatin-activating marks at the GATA2 locus. CRISPRi of the LSD1-bound enhancer in patient-derived TET2 mut AML was associated with dampening of therapeutic GATA2 induction. TET2 knockdown in human hematopoietic stem/progenitor cells induced loss of enhancer 5-hydroxymethylation and facilitated LSD1-mediated enhancer inactivation. Our data provide a basis for rational targeting of cooperating aberrant promoter and enhancer epigenetic marks driven by mutant epigenetic modifiers. SIGNIFICANCE: Somatic mutations of genes encoding epigenetic modifiers are a hallmark of AML and potentially disrupt many components of the epigenome. Our study targets two different epigenetic layers at promoters and enhancers that cooperate to aberrant gene silencing, downstream of the actions of a mutant epigenetic regulator. This article is highlighted in the In This Issue feature, p. 813 ., (©2019 American Association for Cancer Research.)

Clonal hematopoiesis of indeterminate potential (CHIP) is a recently identified process where older patients accumulate distinct subclones defined by recurring somatic mutations in hematopoietic stem cells. CHIP's implications for stem cell transplantation have been harder to identify due to the high degree of mutational heterogeneity that is present within the genetically distinct subclones. In order to gain a better understanding of CHIP and the impact of clonal dynamics on transplantation outcomes, we created a mathematical model of clonal competition dynamics. Our analyses highlight the importance of understanding competition intensity between healthy and mutant clones. Importantly, we highlight the risk that CHIP poses in leading to dominance of precancerous mutant clones and the risk of donor derived leukemia. Furthermore, we estimate the degree of competition intensity and bone marrow niche decline in mice during aging by using our modeling framework. Together, our work highlights the importance of better characterizing the ecological and clonal composition in hematopoietic donor populations at the time of stem cell transplantation., Competing Interests: The authors have declared that no competing interests exist.

Granulocyte-colony stimulating factor receptor (G-CSFR) controls myeloid progenitor proliferation and differentiation to neutrophils. Mutations in CSF3R (encoding G-CSFR) have been reported in patients with chronic neutrophilic leukemia (CNL) and acute myeloid leukemia (AML); however, despite years of research, the malignant downstream signaling of the mutated G-CSFRs is not well understood. Here, we used a quantitative phospho-tyrosine analysis to generate a comprehensive signaling map of G-CSF induced tyrosine phosphorylation in the normal versus mutated (proximal: T618I and truncated: Q741x) G-CSFRs. Unbiased clustering and kinase enrichment analysis identified rapid induction of phospho-proteins associated with endocytosis by the wild type G-CSFR only; while G-CSFR mutants showed abnormal kinetics of canonical Stat3, Stat5, and Mapk phosphorylation, and aberrant activation of Bruton's Tyrosine Kinase (Btk). Mutant-G-CSFR-expressing cells displayed enhanced sensitivity (3-5-fold lower IC50) for ibrutinib-based chemical inhibition of Btk. Primary murine progenitor cells from G-CSFR-Q741x knock-in mice validated activation of Btk by the mutant receptor and retrovirally transduced human CD34 + umbilical cord blood cells expressing mutant receptors displayed enhanced sensitivity to Ibrutinib. A significantly lower clonogenic potential was displayed by both murine and human primary cells expressing mutated receptors upon ibrutinib treatment. Finally, a dramatic synergy was observed between ibrutinib and ruxolinitib at lower dose of the individual drug. Altogether, these data demonstrate the strength of unsupervised proteomics analyses in dissecting oncogenic pathways, and suggest repositioning Ibrutinib for therapy of myeloid leukemia bearing CSF3R mutations. Phospho-tyrosine data are available via ProteomeXchange with identifier PXD009662.

Myelodysplastic syndromes (MDS) are heterogeneous hematopoietic disorders that are incurable with conventional therapy. Their incidence is increasing with global population aging. Although many genetic, epigenetic, splicing, and metabolic aberrations have been identified in patients with MDS, their clinical features are quite similar. Here, we show that hypoxia-independent activation of hypoxia-inducible factor 1α (HIF1A) signaling is both necessary and sufficient to induce dysplastic and cytopenic MDS phenotypes. The HIF1A transcriptional signature is generally activated in MDS patient bone marrow stem/progenitors. Major MDS-associated mutations ( Dnmt3a, Tet2, Asxl1, Runx1 , and Mll1 ) activate the HIF1A signature. Although inducible activation of HIF1A signaling in hematopoietic cells is sufficient to induce MDS phenotypes, both genetic and chemical inhibition of HIF1A signaling rescues MDS phenotypes in a mouse model of MDS. These findings reveal HIF1A as a central pathobiologic mediator of MDS and as an effective therapeutic target for a broad spectrum of patients with MDS. Significance: We showed that dysregulation of HIF1A signaling could generate the clinically relevant diversity of MDS phenotypes by functioning as a signaling funnel for MDS driver mutations. This could resolve the disconnection between genotypes and phenotypes and provide a new clue as to how a variety of driver mutations cause common MDS phenotypes. Cancer Discov; 8(11); 1438-57. ©2018 AACR. See related commentary by Chen and Steidl, p. 1355 This article is highlighted in the In This Issue feature, p. 1333 ., (©2018 American Association for Cancer Research.)

We have shown that antagomiR inhibition of miRNA miR-21 and miR-196b activity is sufficient to ablate MLL-AF9 leukemia stem cells (LSC) in vivo. Here, we used an shRNA screening approach to mimic miRNA activity on experimentally verified miR-196b targets to identify functionally important and therapeutically relevant pathways downstream of oncogenic miRNA in MLL-r AML. We found Cdkn1b (p27 Kip1 ) is a direct miR-196b target whose repression enhanced an embryonic stem cell-like signature associated with decreased leukemia latency and increased numbers of leukemia stem cells in vivo. Conversely, elevation of p27 Kip1 significantly reduced MLL-r leukemia self-renewal, promoted monocytic differentiation of leukemic blasts, and induced cell death. Antagonism of miR-196b activity or pharmacologic inhibition of the Cks1-Skp2-containing SCF E3-ubiquitin ligase complex increased p27 Kip1 and inhibited human AML growth. This work illustrates that understanding oncogenic miRNA target pathways can identify actionable targets in leukemia., (© 2018 Crown copyright. The government of Australia, Canada, or the UK ("the Crown") owns the copyright interests of authors who are government employees. The Crown Copyright is not transferable.)

High-risk B-cell acute lymphoblastic leukemia (B-ALL) remains a therapeutic challenge despite advances in the use of tyrosine kinase inhibitors and chimeric-antigen-receptor engineered T cells. Lymphoblastic-leukemia precursors are highly sensitive to oxidative stress. KLF5 is a member of the Krüppel-like family of transcription factors. KLF5 expression is repressed in B-ALL, including BCR-ABL1+ B-ALL. Here, we demonstrate that forced expression of KLF5 in B-ALL cells bypasses the imatinib resistance which is not associated with mutations of BCR-ABL. Expression of Klf5 impaired leukemogenic activity of BCR-ABL1+ B-cell precursors in vitro and in vivo . The complete genetic loss of Klf5 reduced oxidative stress, increased regeneration of reduced glutathione and decreased apoptosis of leukemic precursors. Klf5 regulation of glutathione levels was mediated by its regulation of glutathione-S-transferase Mu 1 ( Gstm1 ), an important regulator of glutathione-mediated detoxification and protein glutathionylation. Expression of Klf5 or the direct Klf5 target gene Gstm1 inhibited clonogenic activity of Klf5 ∆/∆ leukemic B-cell precursors and unveiled a Klf5-dependent regulatory loop in glutamine-dependent glutathione metabolism. In summary, we describe a novel mechanism of Klf5 B-ALL suppressor activity through its direct role on the metabolism of antioxidant glutathione levels, a crucial positive regulator of leukemic precursor survival., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest.

SET domain containing 2 ( Setd2 ), encoding a histone methyltransferase, is associated with many hematopoietic diseases when mutated. By generating a novel exon 6 conditional knockout mouse model, we describe an essential role of Setd2 in maintaining the adult hematopoietic stem cells. Loss of Setd2 results in leukopenia, anemia, and increased platelets accompanied by hypocellularity, erythroid dysplasia, and mild fibrosis in bone marrow. Setd2 knockout mice show significantly decreased hematopoietic stem and progenitor cells except for erythroid progenitors. Setd2 knockout hematopoietic stem cells fail to establish long-term bone marrow reconstitution after transplantation because of the loss of quiescence, increased apoptosis, and reduced multiple-lineage terminal differentiation potential. Bioinformatic analysis revealed that the hematopoietic stem cells exit from quiescence and commit to differentiation, which lead to hematopoietic stem cell exhaustion. Mechanistically, we attribute an important Setd2 function in murine adult hematopoietic stem cells to the inhibition of the Nsd1/2/3 transcriptional complex, which recruits super elongation complex and controls RNA polymerase II elongation on a subset of target genes, including Myc Our results reveal a critical role of Setd2 in regulating quiescence and differentiation of hematopoietic stem cells through restricting the NSDs/SEC mediated RNA polymerase II elongation., (Copyright© 2018 Ferrata Storti Foundation.)

The advent of facile genome engineering technologies has made the generation of knock-in gene-expression or fusion-protein reporters more tractable. Fluorescent protein labeling of specific genes combined with surface marker profiling can more specifically identify a cell population. However, the question of which fluorescent proteins to utilize to generate reporter constructs is made difficult by the number of candidate proteins and the lack of updated experimental data on newer fluorescent proteins. Compounding this problem, most fluorescent proteins are designed and tested for use in microscopy. To address this, we cloned and characterized the detection sensitivity, spectral overlap, and spillover spreading of 13 monomeric fluorescent proteins to determine utility in multicolor panels. We identified a group of five fluorescent proteins with high signal to noise ratio, minimal spectral overlap, and low spillover spreading making them compatible for multicolor experiments. Specifically, generating reporters with combinations of three of these proteins would allow efficient measurements even at low-level expression. Because the proteins are monomeric, they could function either as gene-expression or as fusion-protein reporters. Additionally, this approach can be generalized as new fluorescent proteins are developed to determine their usefulness in multicolor panels. © 2018 International Society for Advancement of Cytometry., (© 2018 International Society for Advancement of Cytometry.)

Obesity is a chronic organismal stress that disrupts multiple systemic and tissue-specific functions. In this study, we describe the impact of obesity on the activity of the hematopoietic stem cell (HSC) compartment. We show that obesity alters the composition of the HSC compartment and its activity in response to hematopoietic stress. The impact of obesity on HSC function is progressively acquired but persists after weight loss or transplantation into a normal environment. Mechanistically, we establish that the oxidative stress induced by obesity dysregulates the expression of the transcription factor Gfi1 and that increased Gfi1 expression is required for the abnormal HSC function induced by obesity. These results demonstrate that obesity produces durable changes in HSC function and phenotype and that elevation of Gfi1 expression in response to the oxidative environment is a key driver of the altered HSC properties observed in obesity. Altogether, these data provide phenotypic and mechanistic insight into durable hematopoietic dysregulations resulting from obesity., (© 2018 Lee et al.)

Granulocyte-monocyte progenitors (GMPs) and monocyte-dendritic cell progenitors (MDPs) produce monocytes during homeostasis and in response to increased demand during infection. Both progenitor populations are thought to derive from common myeloid progenitors (CMPs), and a hierarchical relationship (CMP-GMP-MDP-monocyte) is presumed to underlie monocyte differentiation. Here, however, we demonstrate that mouse MDPs arose from CMPs independently of GMPs, and that GMPs and MDPs produced monocytes via similar but distinct monocyte-committed progenitors. GMPs and MDPs yielded classical (Ly6C hi ) monocytes with gene expression signatures that were defined by their origins and impacted their function. GMPs produced a subset of "neutrophil-like" monocytes, whereas MDPs gave rise to a subset of monocytes that yielded monocyte-derived dendritic cells. GMPs and MDPs were also independently mobilized to produce specific combinations of myeloid cell types following the injection of microbial components. Thus, the balance of GMP and MDP differentiation shapes the myeloid cell repertoire during homeostasis and following infection., (Copyright © 2017 Elsevier Inc. All rights reserved.)

As a central regulator of cell polarity, the activity of CDC42 GTPase is tightly controlled in maintaining normal hematopoietic stem and progenitor cell (HSC/P) functions. We found that transformation of HSC/P to acute myeloid leukemia (AML) is associated with increased CDC42 expression and activity in leukemia cells. In a mouse model of AML, the loss of Cdc42 abrogates MLL-AF9 -induced AML development. Furthermore, genetic ablation of CDC42 in both murine and human MLL-AF9 (MA9) cells decreased survival and induced differentiation of the clonogenic leukemia-initiating cells. We show that MLL-AF9 leukemia cells maintain cell polarity in the context of elevated Cdc42-guanosine triphosphate activity, similar to nonmalignant, young HSC/Ps. The loss of Cdc42 resulted in a shift to depolarized AML cells that is associated with a decrease in the frequency of symmetric and asymmetric cell divisions producing daughter cells capable of self-renewal. Importantly, we demonstrate that inducible CDC42 suppression in primary human AML cells blocks leukemia progression in a xenograft model. Thus, CDC42 loss suppresses AML cell polarity and division asymmetry, and CDC42 constitutes a useful target to alter leukemia-initiating cell fate for differentiation therapy., (© 2017 by The American Society of Hematology.)

MicroRNA cluster mirn23a has previously been shown to promote myeloid development at the expense of lymphoid development in overexpression and knockout mouse models. This polarization is observed early in hematopoietic development, with an increase in common lymphoid progenitors (CLPs) and a decrease in all myeloid progenitor subsets in adult bone marrow. The pool size of multipotential progenitors (MPPs) is unchanged; however, in this report we observe by flow cytometry that polarized subsets of MPPs are changed in the absence of mirn23a. Additionally, in vitro culture of MPPs and sorted MPP transplants showed that these cells have decreased myeloid and increased lymphoid potential in vitro and in vivo. We investigated the mechanism by which mirn23a regulates hematopoietic differentiation and observed that mirn23a promotes myeloid development of hematopoietic progenitors through regulation of hematopoietic transcription factors and signaling pathways. Early transcription factors that direct the commitment of MPPs to CLPs (Ikzf1, Runx1, Satb1, Bach1 and Bach2) are increased in the absence of mirn23a miRNAs as well as factors that commit the CLP to the B cell lineage (FoxO1, Ebf1, and Pax5). Mirn23a appears to buffer transcription factor levels so that they do not stochastically reach a threshold level to direct differentiation. Intriguingly, mirn23a also inversely regulates the PI3 kinase (PI3K)/Akt and BMP/Smad signaling pathways. Pharmacological inhibitor studies, coupled with dominant active/dominant negative biochemical experiments, show that both signaling pathways are critical to mirn23a's regulation of hematopoietic differentiation. Lastly, consistent with mirn23a being a physiological inhibitor of B cell development, we observed that the essential B cell transcription factor EBF1 represses expression of mirn23a. In summary, our data demonstrates that mirn23a regulates a complex array of transcription and signaling pathways to modulate adult hematopoiesis.

Tyrosine-kinase inhibitor (TKI) therapy for human cancers is not curative, and relapse occurs owing to the continued presence of tumor cells, referred to as minimal residual disease (MRD). The survival of MRD stem or progenitor cells in the absence of oncogenic kinase signaling, a phenomenon referred to as intrinsic resistance, depends on diverse growth factors. Here we report that oncogenic kinase and growth-factor signaling converge to induce the expression of the signaling proteins FBJ osteosarcoma oncogene (c-FOS, encoded by Fos) and dual-specificity phosphatase 1 (DUSP1). Genetic deletion of Fos and Dusp1 suppressed tumor growth in a BCR-ABL fusion protein kinase-induced mouse model of chronic myeloid leukemia (CML). Pharmacological inhibition of c-FOS, DUSP1 and BCR-ABL eradicated MRD in multiple in vivo models, as well as in mice xenotransplanted with patient-derived primary CML cells. Growth-factor signaling also conferred TKI resistance and induced FOS and DUSP1 expression in tumor cells modeling other types of kinase-driven leukemias. Our data demonstrate that c-FOS and DUSP1 expression levels determine the threshold of TKI efficacy, such that growth-factor-induced expression of c-FOS and DUSP1 confers intrinsic resistance to TKI therapy in a wide-ranging set of leukemias, and might represent a unifying Achilles' heel of kinase-driven cancers.

CD8αα TCRαβ + intestinal intraepithelial lymphocytes play a critical role in promoting intestinal homeostasis, although mechanisms controlling their development and peripheral homeostasis remain unclear. In this study, we examined the spatiotemporal role of Bim in the thymic selection of CD8αα precursors and the fate of these cells in the periphery. We found that T cell-specific expression of Bim during early/cortical, but not late/medullary, thymic development controls the agonist selection of CD8αα precursors and limits their private TCRβ repertoire. During this process, agonist-selected double-positive cells lose CD4/8 coreceptor expression and masquerade as double-negative (DN) TCRαβ hi thymocytes. Although these DN thymocytes fail to re-express coreceptors after OP9-DL1 culture, they eventually mature and accumulate in the spleen where TCR and IL-15/STAT5 signaling promotes their conversion to CD8αα cells and their expression of gut-homing receptors. Adoptive transfer of splenic DN cells gives rise to CD8αα cells in the gut, establishing their precursor relationship in vivo. Interestingly, Bim does not restrict the IL-15-driven maturation of CD8αα cells that is critical for intestinal homeostasis. Thus, we found a temporal and tissue-specific role for Bim in limiting thymic agonist selection of CD8αα precursors and their TCRβ repertoire, but not in the maintenance of CD8αα intraepithelial lymphocytes in the intestine., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Delineating hierarchical cellular states, including rare intermediates and the networks of regulatory genes that orchestrate cell-type specification, are continuing challenges for developmental biology. Single-cell RNA sequencing is greatly accelerating such research, given its power to provide comprehensive descriptions of genomic states and their presumptive regulators. Haematopoietic multipotential progenitor cells, as well as bipotential intermediates, manifest mixed-lineage patterns of gene expression at a single-cell level. Such mixed-lineage states may reflect the molecular priming of different developmental potentials by co-expressed alternative-lineage determinants, namely transcription factors. Although a bistable gene regulatory network has been proposed to regulate the specification of either neutrophils or macrophages, the nature of the transition states manifested in vivo, and the underlying dynamics of the cell-fate determinants, have remained elusive. Here we use single-cell RNA sequencing coupled with a new analytic tool, iterative clustering and guide-gene selection, and clonogenic assays to delineate hierarchical genomic and regulatory states that culminate in neutrophil or macrophage specification in mice. We show that this analysis captured prevalent mixed-lineage intermediates that manifested concurrent expression of haematopoietic stem cell/progenitor and myeloid progenitor cell genes. It also revealed rare metastable intermediates that had collapsed the haematopoietic stem cell/progenitor gene expression programme, instead expressing low levels of the myeloid determinants, Irf8 and Gfi1 (refs 9, 10, 11, 12, 13). Genetic perturbations and chromatin immunoprecipitation followed by sequencing revealed Irf8 and Gfi1 as key components of counteracting myeloid-gene-regulatory networks. Combined loss of these two determinants 'trapped' the metastable intermediate. We propose that mixed-lineage states are obligatory during cell-fate specification, manifest differing frequencies because of their dynamic instability and are dictated by counteracting gene-regulatory networks.

Unlabelled: Cytogenetically normal acute myeloid leukemia (CN-AML) represents nearly 50% of human AML. Co-occurring mutations in the de novo DNA methyltransferase DNMT3A and the FMS related tyrosine kinase 3 (FLT3) are common in CN-AML and confer a poorer prognosis. We demonstrate that mice with Flt3-internal tandem duplication (Flt3(ITD)) and inducible deletion of Dnmt3a spontaneously develop a rapidly lethal, completely penetrant, and transplantable AML of normal karyotype. AML cells retain a single Dnmt3a floxed allele, revealing the oncogenic potential of Dnmt3a haploinsufficiency. FLT3(ITD)/DNMT3A-mutant primary human and murine AML exhibit a similar pattern of global DNA methylation associated with changes in the expression of nearby genes. In the murine model, rescuing Dnmt3a expression was accompanied by DNA remethylation and loss of clonogenic potential, suggesting that Dnmt3a-mutant oncogenic effects are reversible. Dissection of the cellular architecture of the AML model using single-cell assays, including single-cell RNA sequencing, identified clonogenic subpopulations that express genes sensitive to the methylation of nearby genomic loci and responsive to DNMT3A levels. Thus, Dnmt3a haploinsufficiency transforms Flt3(ITD) myeloproliferative disease by modulating methylation-sensitive gene expression within a clonogenic AML subpopulation., Significance: DNMT3A haploinsufficiency results in reversible epigenetic alterations that transform FLT3(ITD)-mutant myeloproliferative neoplasm into AML. Cancer Discov; 6(5); 501-15. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 461., (©2016 American Association for Cancer Research.)

Hematopoiesis is characterized by a lifelong balance between hematopoietic stem cell (HSC) self-renewal and differentiation into mature blood populations. Proper instruction of cell fate decisions requires tight homeostatic regulation of transcriptional programs through a combination of epigenetic modifications, management of cis -regulatory elements, and transcription factor activity. Recent work has focused on integrating biochemical, genetic, and evolutionary data sets to gain further insight into these regulatory components. Long noncoding RNA (lncRNA), post-translational modifications of transcription factors, and circadian rhythm add additional layers of complexity. These analyses have provided a wealth of information, much of which has been made available through public databases. Elucidating the regulatory processes that govern hematopoietic transcriptional programs is expected to provide useful insights into hematopoiesis that may be applied broadly across tissue types while enabling the discovery and implementation of therapeutics to treat human disease.

In this issue of Blood, the articles by Shaham et al and Wang et al are the first to identify microRNA 486 (miR-486) as a requisite oncomiR and credible therapeutic target in myeloid leukemia of Down syndrome (ML-DS) and chronic myeloid leukemia (CML) by showing that these 2 leukemias co-opt miR-486 functions in normal erythroid progenitor progrowth and survival activity.

We herein report the design and synthesis of the first nanomolar binding inhibitor of STAT5 protein. Lead compound 13a , possessing a phosphotyrosyl-mimicking salicylic acid group, potently and selectively binds to STAT5 over STAT3, inhibits STAT5-SH2 domain complexation events in vitro , silences activated STAT5 in leukemic cells, as well as STAT5's downstream transcriptional targets, including MYC and MCL1 , and, as a result, leads to apoptosis. We believe 13a represents a useful probe for interrogating STAT5 function in cells as well as being a potential candidate for advanced preclinical trials.

Medulloblastoma is a highly malignant paediatric brain tumour currently treated with a combination of surgery, radiation and chemotherapy, posing a considerable burden of toxicity to the developing child. Genomics has illuminated the extensive intertumoral heterogeneity of medulloblastoma, identifying four distinct molecular subgroups. Group 3 and group 4 subgroup medulloblastomas account for most paediatric cases; yet, oncogenic drivers for these subtypes remain largely unidentified. Here we describe a series of prevalent, highly disparate genomic structural variants, restricted to groups 3 and 4, resulting in specific and mutually exclusive activation of the growth factor independent 1 family proto-oncogenes, GFI1 and GFI1B. Somatic structural variants juxtapose GFI1 or GFI1B coding sequences proximal to active enhancer elements, including super-enhancers, instigating oncogenic activity. Our results, supported by evidence from mouse models, identify GFI1 and GFI1B as prominent medulloblastoma oncogenes and implicate 'enhancer hijacking' as an efficient mechanism driving oncogene activation in a childhood cancer.

Acute myelogenous leukemia (AML) subtypes that result from oncogenic activation of homeobox (HOX) transcription factors are associated with poor prognosis. The HOXA9 transcription activator and growth factor independent 1 (GFI1) transcriptional repressor compete for occupancy at DNA-binding sites for the regulation of common target genes. We exploited this HOXA9 versus GFI1 antagonism to identify the genes encoding microRNA-21 and microRNA-196b as transcriptional targets of HOX-based leukemia oncoproteins. Therapeutic inhibition of microRNA-21 and microRNA-196b inhibited in vitro leukemic colony forming activity and depleted in vivo leukemia-initiating cell activity of HOX-based leukemias, which led to leukemia-free survival in a murine AML model and delayed disease onset in xenograft models. These data establish microRNA as functional effectors of endogenous HOXA9 and HOX-based leukemia oncoproteins, provide a concise in vivo platform to test RNA therapeutics, and suggest therapeutic value for microRNA antagonists in AML.

RUNX1 is generally considered a tumor suppressor in myeloid neoplasms. Inactivating RUNX1 mutations have frequently been found in patients with myelodysplastic syndrome (MDS) and cytogenetically normal acute myeloid leukemia (AML). However, no somatic RUNX1 alteration was found in AMLs with leukemogenic fusion proteins, such as core-binding factor (CBF) leukemia and MLL fusion leukemia, raising the possibility that RUNX1 could actually promote the growth of these leukemia cells. Using normal human cord blood cells and those expressing leukemogenic fusion proteins, we discovered a dual role of RUNX1 in myeloid leukemogenesis. RUNX1 overexpression inhibited the growth of normal cord blood cells by inducing myeloid differentiation, whereas a certain level of RUNX1 activity was required for the growth of AML1-ETO and MLL-AF9 cells. Using a mouse genetic model, we also showed that the combined loss of Runx1/Cbfb inhibited leukemia development induced by MLL-AF9. RUNX2 could compensate for the loss of RUNX1. The survival effect of RUNX1 was mediated by BCL2 in MLL fusion leukemia. Our study unveiled an unexpected prosurvival role for RUNX1 in myeloid leukemogenesis. Inhibiting RUNX1 activity rather than enhancing it could be a promising therapeutic strategy for AMLs with leukemogenic fusion proteins.

Hox genes are key regulators of development. In mammals, the study of these genes is greatly confounded by their large number, overlapping functions and interspersed shared enhancers. Here, we describe the use of a novel recombineering strategy to introduce simultaneous frameshift mutations into the flanking Hoxa9, Hoxa10 and Hoxa11 genes, as well as their paralogs on the HoxD cluster. The resulting Hoxa9,10,11 mutant mice displayed dramatic synergistic homeotic transformations of the reproductive tracts, with the uterus anteriorized towards oviduct and the vas deferens anteriorized towards epididymis. The Hoxa9,10,11 mutant mice also provided a genetic setting that allowed the discovery of Hoxd9,10,11 redundant reproductive tract patterning function. Both shared and distinct Hox functions were defined. Hoxd9,10,11 play a crucial role in the regulation of uterine immune function. Non-coding non-polyadenylated RNAs were among the key Hox targets, with dramatic downregulation in mutants. We observed Hox cross-regulation of transcription and splicing. In addition, we observed a surprising anti-dogmatic apparent posteriorization of the uterine epithelium. In caudal regions of the uterus, the normal simple columnar epithelium flanking the lumen was replaced by a pseudostratified transitional epithelium, normally found near the more posterior cervix. These results identify novel molecular functions of Hox genes in the development of the male and female reproductive tracts.

The 2 main forms of hereditary neutropenia are cyclic (CN) and severe congenital (SCN) neutropenia. CN is an autosomal dominant disorder in which neutrophil counts fluctuate with 21-day periodicity. SCN consists of static neutropenia, with promyelocytic maturation arrest in the bone marrow. Unlike CN, SCN displays frequent acquisition of somatic mutations in the gene CSF3R. CN is caused by heterozygous mutations in the gene ELANE, encoding neutrophil elastase. SCN is genetically heterogeneous but is most frequently associated with ELANE mutations. We discuss how the mutations provide clues into the pathogenesis of neutropenia and describe current hypotheses for its molecular mechanisms., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Growth factor independent 1 (Gfi1) is a transcriptional repressor originally identified as a gene activated in T-cell leukemias induced by Moloney-murine-leukemia virus infection. Notch1 is a transmembrane receptor that is frequently mutated in human T-cell acute lymphoblastic leukemia (T-ALL). Gfi1 is an important factor in the initiation and maintenance of lymphoid leukemias and its deficiency significantly impedes Notch dependent initiation of T-ALL in animal models. Here, we show that immature hematopoietic cells require Gfi1 to competently integrate Notch-activated signaling. Notch1 activation coupled with Gfi1 deficiency early in T-lineage specification leads to a dramatic loss of T-cells, whereas activation in later stages leaves development unaffected. In Gfi1 deficient multipotent precursors, Notch activation induces lethality and is cell autonomous. Further, without Gfi1, multipotent progenitors do not maintain Notch1-activated global expression profiles typical for T-lineage precursors. In agreement with this, we find that both lymphoid-primed multipotent progenitors (LMPP) and early T lineage progenitors (ETP) do not properly form or function in Gfi1(-/-) mice. These defects correlate with an inability of Gfi1(-/-) progenitors to activate lymphoid genes, including IL7R, Rag1, Flt3 and Notch1. Our data indicate that Gfi1 is required for hematopoietic precursors to withstand Notch1 activation and to maintain Notch1 dependent transcriptional programming to determine early T-lymphoid lineage identity., Competing Interests: Dr. H. Leighton Grimes is an Associate Editor at PLOS Genetics. Otherwise, the authors have declared that no relevant competing interests exist.

The coding single nucleotide polymorphism GFI136N in the human gene growth factor independence 1 (GFI1) is present in 3%-7% of whites and increases the risk for acute myeloid leukemia (AML) by 60%. We show here that GFI136N, in contrast to GFI136S, lacks the ability to bind to the Gfi1 target gene that encodes the leukemia-associated transcription factor Hoxa9 and fails to initiate histone modifications that regulate HoxA9 expression. Consistent with this, AML patients heterozygous for the GFI136N variant show increased HOXA9 expression compared with normal controls. Using ChipSeq, we demonstrate that GFI136N specific epigenetic changes are also present in other genes involved in the development of AML. Moreover, granulomonocytic progenitors, a bone marrow subset from which AML can arise in humans and mice, show a proliferative expansion in the presence of the GFI136N variant. In addition, granulomonocytic progenitors carrying the GFI136N variant allele have altered gene expression patterns and differ in their ability to grow after transplantation. Finally, GFI136N can accelerate a K-RAS driven fatal myeloproliferative disease in mice. Our data suggest that the presence of a GFI136N variant allele induces a preleukemic state in myeloid precursors by deregulating the expression of Hoxa9 and other AML-related genes.

One mechanism for disrupting the MLL gene in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) is through partial tandem duplication (MLL-PTD); however, the mechanism by which MLL-PTD contributes to MDS and AML development and maintenance is currently unknown. Herein, we investigated hematopoietic stem/progenitor cell (HSPC) phenotypes of Mll-PTD knock-in mice. Although HSPCs (Lin(-)Sca1(+)Kit(+) (LSK)/SLAM(+) and LSK) in Mll(PTD/WT) mice are reduced in absolute number in steady state because of increased apoptosis, they have a proliferative advantage in colony replating assays, CFU-spleen assays, and competitive transplantation assays over wild-type HSPCs. The Mll(PTD/WT)-derived phenotypic short-term (ST)-HSCs/multipotent progenitors and granulocyte/macrophage progenitors have self-renewal capability, rescuing hematopoiesis by giving rise to long-term repopulating cells in recipient mice with an unexpected myeloid differentiation blockade and lymphoid-lineage bias. However, Mll(PTD/WT) HSPCs never develop leukemia in primary or recipient mice, suggesting that additional genetic and/or epigenetic defects are necessary for full leukemogenic transformation. Thus, the Mll-PTD aberrantly alters HSPCs, enhances self-renewal, causes lineage bias, and blocks myeloid differentiation. These findings provide a framework by which we can ascertain the underlying pathogenic role of MLL-PTD in the clonal evolution of human leukemia, which should facilitate improved therapies and patient outcomes.

MicroRNAs (miRNAs) are highly conserved small RNAs which regulate gene expression primarily through base pairing to the 3' untranslated region of target messenger RNA (mRNA), leading to mRNA degradation or translation inhibition depending on the complementarity between the miRNA and target mRNA. Single miRNA regulates multiple target mRNA. miRNAs have been shown to regulate gene expression in the hematopoietic stem cells, as well as at key decision points for various lineages. However, aberrant expression of miRNAs has been documented in cancer and disease models. Rigorous dissection of miRNA pathways and biology requires facile loss of function modeling. This chapter describes detailed protocol for knockdown miRNA-21 which is involved in myelopoiesis using antagomiRs in primary murine bone marrow stem/progenitor cells.

Protracted inhibition of osteoblast (OB) differentiation characterizes multiple myeloma (MM) bone disease and persists even when patients are in long-term remission. However, the underlying pathophysiology for this prolonged OB suppression is unknown. Therefore, we developed a mouse MM model in which the bone marrow stromal cells (BMSCs) remained unresponsive to OB differentiation signals after removal of MM cells. We found that BMSCs from both MM-bearing mice and MM patients had increased levels of the transcriptional repressor Gfi1 compared with controls and that Gfi1 was a novel transcriptional repressor of the critical OB transcription factor Runx2. Trichostatin-A blocked the effects of Gfi1, suggesting that it induces epigenetic changes in the Runx2 promoter. MM-BMSC cell-cell contact was not required for MM cells to increase Gfi1 and repress Runx2 levels in MC-4 before OBs or naive primary BMSCs, and Gfi1 induction was blocked by anti-TNF-α and anti-IL-7 antibodies. Importantly, BMSCs isolated from Gfi1(-/-) mice were significantly resistant to MM-induced OB suppression. Strikingly, siRNA knockdown of Gfi1 in BMSCs from MM patients significantly restored expression of Runx2 and OB differentiation markers. Thus, Gfi1 may have an important role in prolonged MM-induced OB suppression and provide a new therapeutic target for MM bone disease.

Background & Aims: The tumor suppressors retinoblastoma (RB) and p53 are important regulators of the cell cycle. Although human cancer cells inactivate RB and p53 by many mechanisms, the cooperative roles of these proteins in tumorigenesis are complex and tissue specific. We analyzed the cooperation of RB and p53 in liver development and pathogenesis of hepatocellular carcinoma., Methods: Spontaneous and carcinogen-induced (diethylnitrosamine) tumorigenesis were studied in mice with liver-specific deletions of Rb and/or p53 (Rbf/f;albcre+, p53f/f;albcre+ and Rbf/f; p53f/f;albcre+ mice). Genotype, histologic, immunohistochemical, microarray, quantitative polymerase chain reaction, immunoblot, and comparative genomic hybridization analyses were performed using normal and tumor samples. Comparative microarray analyses were performed against publicly available human microarray data sets., Results: Deletion of RB and p53 from livers of mice deregulated the transcriptional programs associated with human disease. These changes were not sufficient for spontaneous tumorigenesis; potent quiescence mechanisms compensated for loss of these tumor suppressors. In response to hepatocarcinogen-induced damage, distinct and cooperative roles of RB and p53 were revealed; their loss affected cell cycle control, checkpoint response, and genome stability. In damaged tissue, combined loss of RB and p53 resulted in early lesion formation, aggressive tumor progression, and gene expression signatures and histologic characteristics of advanced human hepatocellular carcinoma., Conclusions: The effects RB and p53 loss are determined by the tissue environment; cell stresses that promote aggressive disease reveal the functions of these tumor suppressors., (Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.)

As acute infections resolve, most effector CD8(+) T cells die, whereas some persist and become memory T cells. Recent work showed that subsets of effector CD8(+) T cells, identified by reciprocal expression of killer cell lectin-like receptor G1 (KLRG1) and CD127, have different lifespans. Similar to previous reports, we found that effector CD8(+) T cells reported to have a longer lifespan (i.e., KLRG1(low)CD127(high)) have increased levels of Bcl-2 compared with their shorter-lived KLRG1(high)CD127(low) counterparts. Surprisingly, we found that these effector KLRG1(low)CD127(high) CD8(+) T cells also had increased levels of Bim compared with KLRG1(high)CD127(low) cells. Similar effects were observed in memory cells, in which CD8(+) central memory T cells expressed higher levels of Bim and Bcl-2 than did CD8(+) effector memory T cells. Using both pharmacologic and genetic approaches, we found that survival of both subsets of effector and memory CD8(+) T cells required Bcl-2 to combat the proapoptotic activity of Bim. Interestingly, inhibition or absence of Bcl-2 led to significantly decreased expression of Bim in surviving effector and memory T cells. In addition, manipulation of Bcl-2 levels by IL-7 or IL-15 also affected expression of Bim in effector CD8(+) T cells. Finally, we found that Bim levels were significantly increased in effector CD8(+) T cells lacking Bax and Bak. Together, these data indicate that cells having the highest levels of Bim are selected against during contraction of the response and that Bcl-2 determines the level of Bim that effector and memory T cells can tolerate.

Cdc42 of the Rho GTPase family has been implicated in cell actin organization, proliferation, survival, and migration but its physiological role is likely cell-type specific. By a T cell-specific deletion of Cdc42 in mouse, we have recently shown that Cdc42 maintains naïve T cell homeostasis through promoting cell survival and suppressing T cell activation. Here we have further investigated the involvement of Cdc42 in multiple stages of T cell differentiation. We found that in Cdc42(-/-) thymus, positive selection of CD4(+)CD8(+) double-positive thymocytes was defective, CD4(+) and CD8(+) single-positive thymocytes were impaired in migration and showed an increase in cell apoptosis triggered by anti-CD3/-CD28 antibodies, and thymocytes were hyporesponsive to anti-CD3/-CD28-induced cell proliferation and hyperresponsive to anti-CD3/-CD28-stimulated MAP kinase activation. At the periphery, Cdc42-deficient naive T cells displayed an impaired actin polymerization and TCR clustering during the formation of mature immunological synapse, and showed an enhanced differentiation to Th1 and CD8(+) effector and memory cells in vitro and in vivo. Finally, Cdc42(-/-) mice exhibited exacerbated liver damage in an induced autoimmune disease model. Collectively, these data establish that Cdc42 is critically involved in thymopoiesis and plays a restrictive role in effector and memory T cell differentiation and autoimmunity.

Endotoxin (bacterial lipopolysaccharide [LPS]) causes fatal septic shock via the Toll-like receptor 4 (TLR-4) protein present on innate immunity effector cells, which activates nuclear factor kappa B (NF-kappaB), inducing proinflammatory cytokines, including tumor necrosis factor alpha (TNF-alpha). An early step in this process involves nuclear sequestration of the p65-RelA NF-kappaB subunit, enabling transcriptional activation of target inflammatory cytokine genes. Here, we analyzed the role of the nuclear zinc finger protein Gfi1 in the TLR response using primary bone marrow-derived macrophages. We show that upon LPS stimulation, expression of Gfi1 is induced with kinetics similar to those of nuclear translocation of p65 and that Gfi1 interacts with p65 and inhibits p65-mediated transcriptional transactivation by interfering with p65 binding to target gene promoter DNA. Gfi1-deficient macrophages show abnormally high mRNA levels of the TNF-alpha gene and many other p65 target genes and a higher rate of TNF promoter occupancy by p65 than wild-type cells after LPS stimulation, suggesting that Gfi1 functions as an antagonist of NF-kappaB activity at the level of promoter binding. Our findings identify a new function of Gfi1 as a general negative regulator of the endotoxin-initiated innate immune responses, including septic shock and possibly other severe inflammatory diseases.

Cdc42 is a member of the Rho GTPase family that has been implicated in several cell functions including proliferation and migration, but its physiologic role needs to be dissected in each cell type. We achieved B-cell and hematopoietic stem cell deletion of Cdc42 by conditional gene targeting in mice. Deletion of Cdc42 from proB/preB-cell stage significantly blocked B-cell development at T1 and later stages, resulting in reduced mature B-cell populations and reduced antigen-specific immunoglobulin M (IgM), IgG1, and IgG3 production. The Cdc42(-/-) B cells, themselves, were abnormal with impaired proliferation and survival. The mutant B cells were further characterized by a B-cell receptor (BCR) signaling defect with increased Erk and decreased Akt activation, as well as a defect in BCR-mediated B-cell-activating factor (BAFF) receptor up-regulation and subsequent BAFF receptor signaling in mature resting B cells. Surprisingly, Cdc42 was dispensable for stromal cell-derived factor-1alpha (SDF-1alpha)- or B-lymphocyte chemoattractant (BLC)-induced B-cell migration. Finally, loss of Cdc42 from hematopoietic stem cells did not alter common lymphoid progenitor production but severely reduced proB/preB- and immature B-cell populations, indicating that Cdc42 is also involved in B-cell precursor differentiation. These results reveal multifaceted roles of Cdc42 in B-cell development and activation.

In patients with severe congenital neutropenia (SCN) and mice with growth factor independent-1 (Gfi1) loss of function, arrested myeloid progenitors accumulate, whereas terminal granulopoiesis is blocked. One might assume that Gfi-null progenitors accumulate because they lack the ability to differentiate. Instead, our data indicate that Gfi1 loss of function deregulates 2 separable transcriptional programs, one of which controls the accumulation and lineage specification of myeloid progenitors, but not terminal granulopoiesis. We demonstrate that Gfi1 directly represses HoxA9, Pbx1, and Meis1 during normal myelopoiesis. Gfi1-/- progenitors exhibit elevated levels of HoxA9, Pbx1 and Meis1, exaggerated HoxA9-Pbx1-Meis1 activity, and progenitor transformation in collaboration with oncogenic K-Ras. Limiting HoxA9 alleles corrects, in a dose-dependent manner, in vivo and in vitro phenotypes observed with loss of Gfi1 in myeloid progenitor cells but did not rescue Gfi1-/- blocked granulopoiesis. Thus, Gfi1 integrates 2 events during normal myeloid differentiation; the suppression of a HoxA9-Pbx1-Meis1 progenitor program and the induction of a granulopoietic transcription program.

The zinc finger protein growth factor independent-1 (Gfi1) is a transcriptional repressor that is critically required for normal granulocytic differentiation. GFI1 loss-of-function mutations are found in some patients with severe congenital neutropenia (SCN). The SCN-associated GFI1-mutant proteins act as dominant negatives to block granulopoiesis through selective deregulation of a subset of GFI1 target genes. Here we show that Gfi1 is a master regulator of microRNAs, and that deregulated expression of these microRNAs recapitulates a Gfi1 loss-of-function block to granulocyte colony-stimulating factor (G-CSF)-stimulated granulopoiesis. Specifically, bone marrow cells from a GFI1-mutant SCN patient and Gfi1(-/-) mice display deregulated expression of miR-21 and miR-196B expression. Flow cytometric analysis and colony assays reveal that the overexpression or depletion of either miR induces changes in myeloid development. However, coexpression of miR-21 and miR-196b (as seen in Gfi1(-/-) mice and a GFI1N382S SCN patient) completely blocks G-CSF-induced granulopoiesis. Thus, our results not only identify microRNAs whose regulation is required during myelopoiesis, but also provide an example of synergy in microRNA biologic activity and illustrate potential mechanisms underlying SCN disease pathogenesis.

Lung disease is the major cause of morbidity and mortality in cystic fibrosis, an autosomal recessive disease caused by mutations in CFTR. In cystic fibrosis, chronic infection and dysregulated neutrophilic inflammation lead to progressive airway destruction. The severity of cystic fibrosis lung disease has considerable heritability, independent of CFTR genotype. To identify genetic modifiers, here we performed a genome-wide single nucleotide polymorphism scan in one cohort of cystic fibrosis patients, replicating top candidates in an independent cohort. This approach identified IFRD1 as a modifier of cystic fibrosis lung disease severity. IFRD1 is a histone-deacetylase-dependent transcriptional co-regulator expressed during terminal neutrophil differentiation. Neutrophils, but not macrophages, from Ifrd1-deficient mice showed blunted effector function, associated with decreased NF-kappaB p65 transactivation. In vivo, IFRD1 deficiency caused delayed bacterial clearance from the airway, but also less inflammation and disease-a phenotype primarily dependent on haematopoietic cell expression, or lack of expression, of IFRD1. In humans, IFRD1 polymorphisms were significantly associated with variation in neutrophil effector function. These data indicate that IFRD1 modulates the pathogenesis of cystic fibrosis lung disease through the regulation of neutrophil effector function.

Chromosomal translocations involving the Mixed Lineage Leukemia (MLL) gene produce chimeric proteins that cause abnormal expression of a subset of HOX genes and leukemia development. Here, we show that MLL normally regulates expression of mir-196b, a hematopoietic microRNA located within the HoxA cluster, in a pattern similar to that of the surrounding 5' Hox genes, Hoxa9 and Hoxa10, during embryonic stem (ES) cell differentiation. Within the hematopoietic lineage, mir-196b is most abundant in short-term hematopoietic stem cells and is down-regulated in more differentiated hematopoietic cells. Leukemogenic MLL fusion proteins cause overexpression of mir-196b, while treatment of MLL-AF9 transformed bone marrow cells with mir-196-specific antagomir abrogates their replating potential in methylcellulose. This demonstrates that mir-196b function is necessary for MLL fusion-mediated immortalization. Furthermore, overexpression of mir-196b was found specifically in patients with MLL associated leukemias as determined from analysis of 55 primary leukemia samples. Overexpression of mir-196b in bone marrow progenitor cells leads to increased proliferative capacity and survival, as well as a partial block in differentiation. Our results suggest a mechanism whereby increased expression of mir-196b by MLL fusion proteins significantly contributes to leukemia development.

The growth factor independent 1 (Gfi1) transcriptional regulator oncoprotein plays a crucial role in hematopoietic, inner ear, and pulmonary neuroendocrine cell development and governs cell processes as diverse as self-renewal of hematopoietic stem cells, proliferation, apoptosis, differentiation, cell fate specification, and oncogenesis. However, the molecular basis of its transcriptional functions has remained elusive. Here we show that Gfi1 recruits the histone lysine methyltransferase G9a and the histone deacetylase 1 (HDAC1) in order to modify the chromatin of genes targeted for repression by Gfi1. G9a and HDAC1 are both in a repressive complex assembled by Gfi1. Endogenous Gfi1 colocalizes with G9a, HDAC1, and K9-dimethylated histone H3. Gfi1 associates with G9a and HDAC1 on the promoter of the cell cycle regulator p21Cip/WAF1, resulting in an increase in K9 dimethylation at histone H3. Silencing of Gfi1 expression in myeloid cells reverses G9a and HDAC1 recruitment to p21Cip/WAF1 and elevates its expression. These findings highlight the role of epigenetics in the regulation of development and oncogenesis by Gfi1.

The events that regulate engraftment and long-term repopulating ability of hematopoietic stem cells (HSCs) after transplantation are not well defined. We report for the first time that major histocompatibility complex (MHC) class I K plays a critical role in HSC engraftment via interaction with recipient natural killer (NK) cells. Durable engraftment of purified HSCs requires MHC class I K matching between HSC donor and recipient. In the absence of MHC class I K matching, HSCs exhibit impaired long-term engraftment (P =.01). Dependence on MHC class I K matching is eliminated in B6 beige mice that lack NK cell function, as well as in wild-type mice depleted of NK cells, implicating a possible regulatory role of NK cells for HSC engraftment. The coadministration of CD8+/T-cell receptor-negative (TCR-) graft facilitating cells (FCs) matched at MHC class I K to the HSC donor overcomes the requirement for MHC class I K matching between HSCs and recipient. These data demonstrate that FCs inhibit NK cell effects on the HSCs. Notably, FCs do not suppress the cytotoxic activity of activated NK cells. Enhanced green fluorescent protein-positive (EGFP+) FCs persist for one month following allogeneic transplantation, making cold target inhibition an unlikely mechanism. Therefore, MHC class I may play a critical role in the initiating events that dictate HSC engraftment and/or NK-mediated rejection following allogeneic transplantation.

Growth factor independence-1 (GFI1) and GFI1B are closely related, yet differentially expressed transcriptional repressors with nearly identical DNA binding domains. GFI1 is upregulated in the earliest thymocyte precursors, while GFI1B expression is restricted to T lymphopoiesis stages coincident with activation. Transgenic expression of GFI1 potentiates T-cell activation, while forced GFI1B expression decreases activation. Both mice and humans with mutant Gfi1 display lymphoid abnormalities. Here we describe autoregulation of Gfi1 in primary mouse thymocytes and a human T-cell line. GFI1 binding to cis-element sequences conserved between rat, mouse and human Gfi1 mediates direct and potent transcriptional repression. In addition, dramatic regulation of Gfi1 can also be mediated by GFI1B. These data provide the first example of a gene directly targeted by GFI1 and GFI1B. Moreover, they support a role for auto- and trans-regulation of Gfi1 by GFI1 and GFI1B in maintaining the normal expression patterns of Gfi1, and suggest that GFI1B may indirectly affect T-cell activation through repression of Gfi1.

T cell differentiation in the thymus is dependent upon signaling through the TCR and is characterized by the resulting changes in expression patterns of CD4 and CD8 surface coreceptor molecules. Although recent studies have characterized the effects of proximal TCR signaling on T cell differentiation, the downstream integration of these signals remains largely unknown. The growth factor independence-1 (GFI1) and GFI1B transcriptional repressors may regulate cytokine signaling pathways to affect lymphocyte growth and survival. In this study, we show that Gfi1 expression is induced upon induction of the T cell program. Gfi1B expression is low and dynamic during T cell development, but is terminated in mature thymocytes. Transgenic expression of GFI1 and GFI1B in T cells allowed us to determine the functional consequences of constitutive expression. GFI1 potentiates response to TCR stimulation and IL-2, whereas GFI1B-transgenic T cells are defective in T cell activation. Moreover, GFI1B-transgenic thymocytes display reduced expression of the late-activation marker IL-7R alpha, and a decrease in CD4(-)8(+) single-positive T cells that can be mitigated by transgenic expression of BCL2 or GFI1. These data show that GFI1 and GFI1B are functionally unique, and implicate a role for GFI1 in the integration of activation and survival signals.

Gfi1 was first identified as causing interleukin 2-independent growth in T cells and lymphomagenesis in mice. Much work has shown that Gfi1 and Gfi1b, a second mouse homolog, play pivotal roles in blood cell lineage differentiation. However, neither Gfi1 nor Gfi1b has been implicated in nervous system development, even though their invertebrate homologues, senseless in Drosophila and pag-3 in C. elegans are expressed and required in the nervous system. We show that Gfi1 mRNA is expressed in many areas that give rise to neuronal cells during embryonic development in mouse, and that Gfi1 protein has a more restricted expression pattern. By E12.5 Gfi1 mRNA is expressed in both the CNS and PNS as well as in many sensory epithelia including the developing inner ear epithelia. At later developmental stages, Gfi1 expression in the ear is refined to the hair cells and neurons throughout the inner ear. Gfi1 protein is expressed in a more restricted pattern in specialized sensory cells of the PNS, including the eye, presumptive Merkel cells, the lung and hair cells of the inner ear. Gfi1 mutant mice display behavioral defects that are consistent with inner ear anomalies, as they are ataxic, circle, display head tilting behavior and do not respond to noise. They have a unique inner ear phenotype in that the vestibular and cochlear hair cells are differentially affected. Although Gfi1-deficient mice initially specify inner ear hair cells, these hair cells are disorganized in both the vestibule and cochlea. The outer hair cells of the cochlea are improperly innervated and express neuronal markers that are not normally expressed in these cells. Furthermore, Gfi1 mutant mice lose all cochlear hair cells just prior to and soon after birth through apoptosis. Finally, by five months of age there is also a dramatic reduction in the number of cochlear neurons. Hence, Gfi1 is expressed in the developing nervous system, is required for inner ear hair cell differentiation, and its loss causes programmed cell death.