Role In Lineage Determination Research Articles

SMAD4 and TGFβ are architects of inverse genetic programs during fate determination of antiviral CTLs.

Transforming growth factor β (TGFβ) is an important differentiation factor for cytotoxic T lymphocytes (CTLs) and alters the expression levels of several of homing receptors during infection. SMAD4 is part of the canonical signaling network used by members of the transforming growth factor family. For this study, genetically modified mice were used to determine how SMAD4 and TGFβ receptor II (TGFβRII) participate in transcriptional programming of pathogen-specific CTLs. We show that these molecules are essential components of opposing signaling mechanisms, and cooperatively regulate a collection of genes that determine whether specialized populations of pathogen-specific CTLs circulate around the body, or settle in peripheral tissues. TGFβ uses a canonical SMAD-dependent signaling pathway to downregulate Eomesodermin (EOMES), KLRG1, and CD62L, while CD103 is induced. Conversely, in vivo and in vitro data show that EOMES, KLRG1, CX3CR1, and CD62L are positively regulated via SMAD4, while CD103 and Hobit are downregulated. Intravascular staining also shows that signaling via SMAD4 promotes formation of long-lived terminally differentiated CTLs that localize in the vasculature. Our data show that inflammatory molecules play a key role in lineage determination of pathogen-specific CTLs, and use SMAD-dependent signaling to alter the expression levels of multiple homing receptors and transcription factors with known functions during memory formation.

FOXO1 Is Present in Stomach Epithelium and Determines Gastric Cell Distribution.

BACKGROUND AND AIMS:Stomach cells can be converted to insulin-producing cells by Neurog3, MafA, and Pdxl over-expression. Enteroendocrine cells can be similarly made to produce insulin by the deletion of FOXO1. Characteristics and functional properties of FOXO1-expressing stomach cells are not known.METHODS:Using mice bearing a FOXO1-GFP knock-in allele and primary cell cultures, we examined the identity of FOXO1-expressing stomach cells and analyzed their features through loss-of-function studies with red-to-green fluorescent reporters.RESULTS:FOXO1 localizes to a subset of Neurog3 and parietal cells. FOXO1 deletion ex vivo or in vivo using Neurog3-cre or Atp4b-cre increased numbers of parietal cells, generated insulin- and C-peptide-immunoreactive cells, and raised Neurog3 messenger RNA. Gene expression and ChIP- seq experiments identified the cell cycle regulator cyclin E1 (CCNE1) as a FOXO1 target.CONCLUSION:FOXO1 is expressed in a subset of stomach cells. Its ablation increases parietal cells and yields insulin-immunoreactive cells, consistent with a role in lineage determination.

Understanding Erythropoiesis Using Quantitative Proteomics and Single Cell Mass Cytometry

The process of erythropoiesis whereby hematopoietic stem cells (HSC) differentiate into cells with increasingly restricted potential is regulated by a network of lineage-specifying transcription factors (LS-TFs) that promote erythropoiesis while simultaneously repressing other hematopoietic lineages. While TFs that stimulate differentiation towards the erythroid lineage (such as GATA1 and KLF1/EKLF) are more abundant in erythroid progenitors and precursors, these proteins are also expressed in hematopoietic stem/progenitor cells (HSPCs) although at very low levels. This suggests that the dosage of TFs plays an important role in lineage determination. Consistent with this, previous lineage reprogramming experiments have shown that ectopic expression of the same LS-TF can give rise to distinct cell fates depending on the TF's abundance. Furthermore, a controversial model proposed that TFs belonging to competing hematopoietic lineages are co-expressed in bipotential progenitors, and that changes in their relative levels drive differentiation towards one fate or another. Taken together, this suggests that changes in LS-TFs stoichiometry may be central to cell fate choice and lineage commitment. While gene regulatory networks have been established to model the process of cell fate decision in bipotential progenitors, these network models are based on mRNA measurements and have not integrated protein levels of TFs. This is a problem as protein levels do not always correlate with mRNA levels, and as such the gene regulatory network underlying erythroid lineage determination is currently unclear. While standard proteomic approaches such as Western blot or data-dependent mass spectrometry (i.e. shotgun mass spectrometry) are useful to measure changes in the relative level of a single protein over time, these approaches do not provide information on the relative levels between several proteins in the same sample, and as such, changes in protein stoichiometry for master regulators of erythropoiesis remain mostly unknown. To address these questions and to provide a better understanding of the role and importance of quantitative changes in LS-TFs for the process of erythroid lineage commitment, we have used a combination of single cell proteomic (i.e. mass cytometry or CyTOF) and targeted mass spectrometry (i.e. SRM for selected reaction monitoring coupled with the spiking of known amounts of internal standard peptides) approaches to measure changes in protein levels of master regulators of hematopoiesis and erythropoiesis. As a model system for human erythropoiesis, we used cord-blood derived CD34+ HSPCs that are driven to differentiate along the erythroid lineage using a combination of growth factors and cytokines 1. Cells were harvested every second day from HSPCs to differentiated erythroid cells. For CyTOF analysis, cells were barcoded at each time-point, combined and stained with a cocktail of antibodies against 11 cell surface markers and 16 TFs. Clustering analysis was then used to establish a temporal trajectory of erythropoiesis. The data revealed that competing LS-TFs proteins (e.g. KLF1 a pro-erythroid factor and FLI1 a pro-megakaryocyte factor) are co-expressed in bipotential progenitors at equimolar levels. Furthermore, relative levels of KLF1 vs FLI1 change gradually as the cells progress along the erythroid trajectory. Finally, ectopic expression of FLI1 in early progenitors actively deviates cells from their preferred erythroid trajectory towards a megakaryocytic lineage 2. Thus, our results support the concept that temporally-regulated quantitative changes in TFs protein levels in individual hematopoietic progenitors are key determinants of cell fate decision in human erythropoiesis. Current studies are ongoing to identify additional pairs of LS-TFs that regulate other hematopoietic transitions. Furthermore, a dynamic gene regulatory network of erythroid lineage commitment that integrates protein and mRNA data for master regulators of hematopoiesis has been established. Giarratana MC, Kobari L, Lapillonne H, et al. Ex vivo generation of fully mature human red blood cells from hematopoietic stem cells. Nat Biotechnol. 2005;23(1):69-74. Palii CG, Cheng Q, Gillespie MA, et al. Single-Cell Proteomics Reveal that Quantitative Changes in Co-expressed Lineage-Specific Transcription Factors Determine Cell Fate. Cell Stem Cell. 2019;24(5):812-820 e815. Disclosures No relevant conflicts of interest to declare.

Abstract B35: Runx3 contributes for the defense against endogenous level of oncogenic K-Ras-induced tumorigenesis

Abstract Recent studies demonstrated that p53 affects only adenocarcinomas in K-RasG12V knockin mice leaving untouched adenomas which are likely to progress into adenocarcinomas (1, 2). These observations raised a fundamental question regarding whether cells can defend against endogenous level of oncogenic signal (3). Runx3, which plays pivotal roles in lineage determination and functions as a tumor suppressor, is frequently inactivated by promoter hypermethylation in various types of human tumors (4). Previously, we showed that deletion of Runx3 markedly enhanced K-Ras-G12V-induced lung tumorigenesis and led to early death (5). In this study, we found that immortalized Runx3-/- mouse embryonic fibroblast cells (MEFs) were tumorigenic. In addition, Runx3 was silenced by promoter hypermethylation in K-Ras-G12V-knockin MEFs. Expression of Runx3 in the K-Ras-G12V MEFs abolished their tumorigenesity. In mice, knockin of K-Ras-G12V in limited number of cells failed to induce tumor, but combination of K-Ras-G12V-knockin and Runx3-knockout by the same targeting method rapidly induced severe lung adenocarcinomas, lymphomas and skin carcinomas (median survival, 48 days). RUNX3 was silenced in all cases of K-Ras-activated human non-small cell lung cancers (n=20). Our results demonstrate that mammalian cells can defend against endogenous oncogenic K-Ras and Runx3 plays a key role in the defense mechanism.

Expression of the new P2Y-like receptor GPR17 during oligodendrocyte precursor cell maturation regulates sensitivity to ATP-induced death

The P2Y-like receptor GPR17 is expressed by adult neural progenitor cells, suggesting a role in lineage determination. Here, we characterized GPR17 expression and function in mouse cortical primary astrocytes/precursor cell cultures. GPR17 is expressed by a subpopulation of oligodendrocyte precursor cells (OPCs), but not by astrocytes. This expression pattern was also confirmed in vivo. In vitro, GPR17 expression was markedly influenced by culturing conditions. In the presence of growth factors (GFs), no significant GPR17 expression was found. When cultures were shifted to a differentiating medium, a dramatic, time-dependent increase in the number of highly branched GPR17-positive cells was observed. Under these conditions, GPR17 was induced in the totality of O4-positive immature oligodendrocytes. Instead, in cultures originally grown in the absence of GFs, GPR17 was already expressed in morphologically more mature OPCs. Shifting of these cultures to differentiating conditions induced GPR17 only in a subpopulation of O4-positive cells. Under both culture protocols, appearance of more mature CNPase- and MBP-positive cells was associated to a progressive loss of GPR17. GPR17 expression also sensitized cells to adenine nucleotide-induced cytotoxicity, whereas activation with uracil nucleotides promoted differentiation towards a more mature phenotype. We suggest that GFs may keep OPCs in a less differentiated stage by restraining GPR17 expression, and that, under permissive conditions, GPR17 contributes to OPCs differentiation. However, upon high extracellular adenine nucleotide concentrations, as during trauma and ischemia, GPR17 sensitizes cells to cytotoxicity. This double-edged sword role may be exploited to unveil new therapeutic approaches to acute and chronic brain disorders.

Tcf3 and Tcf4 are essential for long-term homeostasis of skin epithelia

Single-layered embryonic skin either stratifies to form epidermis or responds to Wnt signaling (stabilized beta-catenin) to form hair follicles. Postnatally, stem cells continue to differentially use Wnt signaling in long-term tissue homeostasis. We have discovered that embryonic progenitor cells and postnatal hair follicle stem cells coexpress Tcf3 and Tcf4, which can act as transcriptional activators or repressors. Using loss-of-function studies and transcriptional analyses, we uncovered consequences to the absence of Tcf3 and Tcf4 in skin that only partially overlap with those caused by beta-catenin deficiency. We established roles for Tcf3 and Tcf4 in long-term maintenance and wound repair of both epidermis and hair follicles, suggesting that Tcf proteins have both Wnt-dependent and Wnt-independent roles in lineage determination.

Expression of the endocannabinoid system in the bi-potential HEL cell line: commitment to the megakaryoblastic lineage by 2-arachidonoylglycerol

The role of the endocannabinoid system in haematopoietic cells is not completely understood. We investigated whether human erythroleukemia (HEL) cells were able to bind, metabolise and transport the main endocannabinoids, anandamide (AEA) and 2-arachidonoylglycerol (2-AG). We also investigated whether AEA or 2-AG could modulate HEL differentiation. Although able to internalise both endocannabinoids, HEL cells had the machinery to metabolise 2-AG only, since they were devoid of the enzymes needed to synthesise and degrade AEA. Nonetheless, the intracellular transport of exogenous AEA might be required to activate the vanilloid receptors, with yet unknown implications for vascular biology. On the contrary, 2-AG appeared to play a role in lineage determination. Indeed, 2-AG itself drove HEL cells towards megakaryocytic differentiation, as it enhanced expression of beta3 integrin subunit, a megakaryocyte/platelet surface antigen, and glycoprotein VI, a late marker of megakaryocytes; in parallel, it reduced the amount of messenger RNA encoding for glycophorin A, a marker of erythroid phenotype. All these effects were mediated by activation of CB(2) cannabinoid receptors that triggered an extracellular signal-regulated kinase-dependent signalling cascade. In addition, classical inducers of megakaryocyte differentiation reduced 2-AG synthesis (although they did not affect the binding efficiency of CB(2) receptors), suggesting that levels of this endocannabinoid may be critical for committing HEL cells towards the megakaryocytic lineage.

T-box proteins differentially activate the expression of the endogenous interferon γ gene versus transfected reporter genes in non-immune cells

The T-box transcription factor T-bet is expressed in a number of hematopoetic cell types and plays an essential role in the lineage determination of Th1 T-helper cells. In the absence of T-bet, CD4 + T-cells fail to induce IFNγ, the cytokine whose expression characterizes Th1 cells. Here we show that, surprisingly, T-bet induces the expression of endogenous IFNγ in non-immune human cells, including 293 and other cell lines. Thus T-bet can induce IFNγ expression independently of its role in T-cell lineage determination. In addition, mutations in T-bet, and chimeras of T-bet with other transcription factors including the T-box transcription factor, TBX2, differentially affect the ability of T-bet to activate expression of endogenous IFNγ versus a T-site regulated reporter gene. A truncated T-betVp16 fusion protein strongly activates the T-site reporter but fails to activate endogenous IFNγ. Conversely, native T-bet strongly activates endogenous IFNγ expression but only weakly activates the reporter gene. Fusion of the Vp16 activation domain to full-length T-bet greatly increases its activation of both endogenous IFNγ and transfected T-site reporter gene expression. In contrast, TBX2Vp16 potently activates the T-site reporter but has a negligible effect on endogenous IFNγ expression. Butyrate treatment of T-bet expressing cells potentiates the expression of endogenous IFNγ but weakly represses expression of the T-site reporter gene. These data indicate that induction of endogenous IFNγ can be uncoupled from differentiation into the Th1 lineage and that the expression of endogenous IFNγ versus a T-site reporter gene is differentially regulated by T-bet and other T-box proteins.

TCR Signal Strength Influences αβ/γδ Lineage Fate

Signals transduced by T cell antigen receptors (TCRs) have been shown to be critical for alphabeta and gammadelta T cell development, but their role in lineage determination remains poorly defined. Two models have been forwarded for alphabeta/gammadelta lineage choice: the instructive model and the stochastic model. Recent data, however, are inconsistent with either model. In this study, we devised an experimental system in which lineage fate was controlled exclusively by the gammadeltaTCR. We then analyzed the impact of TCR signal strength on alphabeta/gammadelta lineage development by altering the surface expression or signaling potential of the gammadeltaTCR complex. We found that increasing the gammadeltaTCR signal strength favored gammadelta lineage development, whereas weakening the gammadeltaTCR signal favored alphabeta lineage development. These results support a model in which the strength of the TCR signal is a critical determinant in the lineage fate decision.

BCL-2 and BCL-XL Restrict Lineage Choice during Hematopoietic Differentiation

Differentiation of hematopoietic cells from multipotential progenitors is regulated by multiple growth factors and cytokines. A prominent feature of these soluble factors is promotion of cell survival, in part mediated by expression of either of the anti-apoptotic proteins, BCL-2 and BCL-XL. The complex expression pattern of these frequently redundant survival factors during hematopoiesis may indicate a role in lineage determination. To investigate the latter possibility, we analyzed factor-dependent cell-Patersen (FDCP)-Mix multipotent progenitor cells in which we stably expressed BCL-2 or BCL-XL. Each factor maintained complete survival of interleukin-3 (IL-3)-deprived FDCP-Mix cells but, unexpectedly, directed FDCP-Mix cells along restricted and divergent differentiation pathways. Thus, IL-3-deprived FDCP-Mix BCL-2 cells differentiated exclusively to granulocytes and monocytes/macrophages, whereas FDCP-Mix BCL-XL cells became erythroid. FDCP-Mix BCL-2 cells grown in IL-3 were distinguished from FDCP-Mix and FDCP-Mix BCL-XL cells by a striking reduction in cellular levels of Raf-1 protein. Replacement of the BCL-2 BH4 domain with the related BCL-XL BH4 sequence resulted in a switch of FDCP-Mix BCL-2 cells to erythroid fate accompanied by persistence of Raf-1 protein expression. Moreover, enforced expression of Raf-1 redirected FDCP-Mix BCL-2 cells to an erythroid fate, and prohibited generation of myeloid cells. These results identify novel roles for BCL-2 and BCL-XL in cell fate decisions beyond cell survival. These effects are associated with differential regulation of Raf-1 expression, perhaps involving the previously identified interaction between BCL-2-BH4 and the catalytic domain of Raf-1.

Identification of an Osteoclast Transcription Factor That Binds to the Human T Cell Leukemia Virus Type I-Long Terminal Repeat Enhancer Element

Transgenic mice expressing human T cell leukemia virus type I (HTLV-I)-tax under the control of HTLV-I-long terminal repeat (LTR) promoter develop skeletal abnormalities with high bone turnover and myelofibrosis. In these animals, Tax is highly expressed in bone with a pattern of expression restricted to osteoclasts and spindle-shaped cells within the endosteal myelofibrosis. To test the hypothesis that lineage-specific transcription factors promote transgene expression from the HTLV-I-LTR in osteoclasts, we first examined tax expression in transgenic bone marrow cultures. Expression was dependent on 1alpha,25-dihydroxycholecalciferol and coincided with tartrate-resistant acid phosphatase (TRAP) expression, a marker of osteoclast differentiation. Furthermore, Tax was expressed in vitronectin receptor-positive mononuclear precursors as well as in mature osteoclast-like cells (OCLs). Consistent with our hypothesis, electrophoretic mobility shift assays revealed the presence of an OCL nuclear factor (NFOC-1) that binds to the LTR 21-base pair direct repeat, a region critical for the promoter activity. This binding is further enhanced by Tax. Since NFOC-1 is absent in macrophages and conserved in osteoclasts among species including human, such a factor may play a role in lineage determination and/or in expression of the differentiated osteoclast phenotype.

Protein kinase C in erythroid and megakaryocytic differentiation: possible role in lineage determination

Erythroid differentiation of normal human hematopoietic progenitor cells was drastically inhibited by phorbol ester, 12-myristate 13-acetate (PMA), an agent known to activate the class of serine-threonine kinases, protein kinase C (PKC). This inhibition was accompanied by augmented megakaryocytic differentiation as demonstrated by expression of megakaryocyte-specific mRNAs and proteins. These effects of PMA were reversed by two specific antagonists of PKC. Analysis of single colonies transferred from cultures not containing PMA to PMA-containing cultures indicated that, in this system, PMA exerts megakaryocytic differentiating activity directly on cells which may have already initiated a progression toward the erythroid pathway of differentiation. These results suggest that modulation of PKC activity plays a role in erythroid and megakaryocytic differentiation, and may constitute an important selective signal between these pathways during normal blood cell development.

Transcription Factors and Hematopoietic Development

Transcription Factors and Hematopoietic Development

L- myc and N- myc influence lineage determination in the central nervous system

The N- myc and the L- myc proto-oncogenes are expressed during embryonal development mainly in the developing brain. Studies of their expression in single neuroepithelial cells revealed that neural precursors not yet committed to the glial or the neuronal lineage expressed both genes, but after lineage commitment they expressed either N- myc or L- myc. Moreover, enforced expression of L- myc in the neural precursor cell line 2.3D caused neuronal differentiation, while the expression of N- myc promoted glial differentiation. These results indicate that L- myc and N- myc play critical roles in lineage determination for the central nervous system.

Coordinate regulation of HOX genes in human hematopoietic cells.

Hematopoiesis is a continuous process in which precursor cells proliferate and differentiate throughout life. However, the molecular mechanisms that govern this process are not clearly defined. Homeobox-containing genes, encoding DNA-binding homeodomains, are a network of genes highly conserved throughout evolution. They are organized in clusters expressed in the developing embryo with a positional hierarchy. We have analyzed expression of the four human HOX loci in erythroleukemic, promyelocytic, and monocytic cell lines to investigate whether the physical organization of human HOX genes reflects a regulatory hierarchy involved in the differentiation process of hematopoietic cells. Our results demonstrate that cells representing various stages of hematopoietic differentiation display differential patterns of HOX gene expression and that HOX genes are coordinately switched on or off in blocks that may include entire loci. The entire HOX4 locus is silent in all lines analyzed and almost all the HOX2 genes are active in erythroleukemic cells and turned off in myeloid-restricted cells. Our observations provide information about the regulation of HOX genes and suggest that the coordinate regulation of these genes may play an important role in lineage determination during early steps of hematopoiesis.