Inferring rules of lineage commitment in haematopoiesis
ABSTRACT: Fractionation of EML cells isolated based on surface expression of immunophenotypic markers reveals the existence of a subpopulation that has undergone spontaneous commitment to an erythroid fate. Uncommitted fractions were compared to committed cells, derived from both spontaneous (EryCP) and cytokine-driven (Ediff) commitment. EML cells were separated by flow cytometry based on their expression of the markers Sca1, CD34 and cKit as described.
Project description:During commitment of a multipotent stem or progenitor cell to a particular lineage, a large number of genes alter their expression in a coordinated manner orchestrated by the gene regulatory network (GRN). The constraints imposed by the GRN govern how cells move in the high-dimensional gene expression state space and can be understood as a dynamical system in which phenotypic cell states (cell types) are attractors that stabilize the cell-type characteristic gene expression pattern against molecular noise. Despite insights from various theoretical models, it remains elusive how multipotent cells, when committing to a specific lineage, exit their attractor and enter a new distinct attractor. Here we show, using single-cell resolution monitoring of transcript patterns by qPCR that commitment of multipotent blood progenitor cells to either the erythroid or the myeloid lineage is preceded by a destabilization of the progenitors’ attractor state and a slowing-down of relaxation of cells from outlier states, indicating a critical state transition (“tipping point”). The high-dimensionality of the system (many genes) and availability of individual trajectories of a large ensemble of systems (many cells) affords a novel signature for critical transition which can be predicted from theory: Decrease of correlation between cells and concomitant increase of correlation between genes as the cell population approaches the tipping point. Consistent with a destabilizing bifurcation that simultaneously opens access to the erythroid and myeloid attractors, differentiation signal for either lineage caused some cells to commit to the “wrong” fate; moreover providing conflicting signals resulted in a delayed decision at the bifurcation point that however was ultimately resolved by commitment to one fate. These results suggest that the theoretical framework of “early-warning signs” and critical transitions can be applied to ensembles of high-dimensional systems, offering a formal tool for analyzing single-cell omics data beyond current descriptive computational pattern recognition. Mouse blood progenitor cells (EML cell line) was exposed to EPO, IL-3/GM-CSF or a mixture of both cytokines and gene expression change was measured in sorted subpopulations wrt Sca1 progenitor surface marker expression. In total, there was 4 conditions (including control), three time points (including d0) and 20 samples (10 samples in duplicates) were analyzed. Two independent experiments were performed for each condition. The untreated progenitor cell population was used as control.
Project description:ASXL1 is the obligate regulatory subunit of a deubiquitinase complex whose catalytic subunit is BAP1. Heterozygous mutations of ASXL1 that result in premature truncations are frequent in myeloid leukemias and Bohring-Opitz syndrome. Here, we demonstrate that truncated ASXL1 proteins confer enhanced activity on the ASXL1-BAP1 complex. Stable expression of truncated, hyperactive ASXL1-BAP1 complexes in a hematopoietic precursor cell line resulted in global erasure of H2AK119Ub, striking depletion of H3K27me3, selective upregulation of a subset of genes whose promoters bore both H2AK119Ub and H3K4me3, and spontaneous differentiation to the mast cell lineage. These outcomes required the catalytic activity of BAP1, indicating these events were downstream consequences of H2AK119Ub erasure. In bone marrow precursors, truncated ASXL1-BAP1 expression cooperated with TET2 loss-of-function to increase differentiation to the myeloid lineage in vivo. We propose that pathological ASXL1 mutations confer gain-of-function on the ASXL-BAP1 complex. ChIP-Seq for H2AK119Ub, H3K4me3, H3K27me3 on EML cells. RNA-Seq on EML cells expressing ASXL1(1-479)+BAP1 and control.
Project description:Enforced expression of the homeobox transcription factor HOXB4 has been shown to enhance hematopoietic stem cell (HSC) self-renewal and expansion ex vivo and in vivo. In order to investigate the largely unknown downstream targets of HOXB4 in hematopoietic progenitor cells, HOXB4 was constitutively overexpressed in the primitive hematopoietic progenitor cell line, EML. Gene expression differences were compared between KLS (c-Kit+, Lin-, Sca-1+)-EML cells that overexpressed HOXB4 (KLS-EML-HOXB4) to control KLS-EML cells that were transduced with vector alone. ChIP-chip was used to identify promoter regions bound by HOXB4. We overexpressed HOXB4 in EML cells. We isolated 3 separate single cell clones as assessed by Southern Blot Analysis (3 clones for EML-HOXB4 and 3 clones for control EML-GFP cells). RNA was isolated from the KLS (c-Kit+, Lin-, Sca-1+) fraction of each single cell clone population and processed for hybridization to array chips using established lab protocols. Chip-Chip analysis of the three HOXB4 overexpressing clones was performed to identify HOXB4 bound promoters.
Project description:We sequenced mRNA from mouse EML cell line under Kai1 knockdown or Kai1 overexpressing condition. KAI1 belongs to tetraspanin superfamily and is known as suppressor of tumor metastasis. Examination of mRNA levels in wildtype (WT) and KAI1 knockdown EML cells were generated by deep sequencing, in duplicate, using Illumina GAIIx. Examination of mRNA levels in wildtype (WT) and KAI1 overexpressing EML cells were also generated in single set.
Project description:Most adult patients have a D816V mutation in phosphotransferase domain (PTD), we have described that half of the children carry mutations in extracellular domain (ECD). KIT-ECD versus IT-PTD-mutants were introduced into rodent Ba/F3, EML, Rat2 and human TF1 cells to investigate their biological effect. ECD- and PTD-mutants also displayed distinct whole-genome transcriptional profiles in EML cells. We observed differences in their signaling properties: they both activated STAT pathways, whereas AKT pathway was only activated by ECD-mutants. Consistently, AKT inhibitor suppressed ECD-mutant-dependent proliferation, clonogenicity and erythroid differentiation. Expression of myristoylated AKT restored erythroid differentiation in EMLPTD cells, suggesting the differential role of AKT in those mutants. Overall, our study implied different pathogenesis of pediatric versus adult mastocytosis, which might explain their diverse phenotypes. Each EML cell line (Del417-419insY mutant and D816V mutant) was cultured with or without 250 ng/mL SCF for 48h. Gene expression profile analysis was performed using whole-genome microarrays. RNA expression profiling of cell lines was done with Affymetrix M430 2.0 mouse oligonucleotide microarrays containing 45.101 probe sets, representing 21.408 transcripts and variants including 17.482 well-characterized mouse genes. Preparation of cRNA, hybridizations, washes, detection and quantification were done as recommended by the supplier. For each sample, synthesis of the first-strand cDNA was done from 3 μg total RNA by T7-oligo(dT) priming, followed by second-strand cDNA synthesis. After purification, in vitro transcription associated with amplification generated cRNA-containing biotinylated pseudouridine. Biotinylated cRNA was purified, quantified and chemically fragmented (95°C for 35 min), then hybridized to microarrays in 200 μL hybridization buffer at 45°C for 16 h. Automated washes and staining with streptavidin-phycoerythrin were done as recommended. Signal amplification was done by biotinylated antistreptavidin antibody with goat-IgG blocking antibody. Scanning was done with Affymetrix GeneArray scanner and quantification with Affymetrix GCOS software.
Project description:A critical problem in biology is understanding how cells choose between self-renewal and differentiation. To generate a comprehensive view of the mechanisms controlling early hematopoietic precursor self-renewal and differentiation, we used systems-based approaches and murine EML multipotential hematopoietic precursor cells as a primary model. EML cells give rise to a mixture of self-renewing Lin-SCA+CD34+ cells and partially differentiated non-renewing Lin-SCA-CD34- cells in a cell autonomous fashion. We identified and validated the HMG box protein TCF7 as a key regulator in this self-renewal/differentiation switch, and it operates in the absence of canonical Wnt signaling. We found that TCF7 is the most downregulated transcription factor when CD34+ cells switch into CD34- cells using RNA-Seq. We subsequently identified the target genes bound by TCF7 using ChIP-Seq. We show that TCF7 binds to Runx1 (Aml1) promoter region, and RUNX1 and TCF7 co-regulate. Gene Set Enrichment Analysis suggests that TCF7 primarily acts as a positive regulator of genes preferentially expressed in CD34+ cells. Consistent with this possibility, knocking-down TCF7 represses many up-regulated genes in Lin-CD34+ cells. Finally a network of up-regulated transcription factors of CD34+ cells which defines the self-renewing state was constructed. These studies in EML cells demonstrate fundamental cell-intrinsic properties of the switch between self-renewal and differentiation, and yield valuable insights for manipulating HSCs and other differentiating systems. The gene expression changes when TCF7 is knocked-down by shRNA in Lin-SCA+CD34+ cells were identified by microarray analysis of one control and two experimental samples.
Project description:A critical problem in biology is understanding how cells choose between self-renewal and differentiation. To generate a comprehensive view of the mechanisms controlling early hematopoietic precursor self-renewal and differentiation, we used systems-based approaches and murine EML multipotential hematopoietic precursor cells as a primary model. EML cells give rise to a mixture of self-renewing Lin-SCA+CD34+ cells and partially differentiated non-renewing Lin-SCA-CD34- cells in a cell autonomous fashion. We identified and validated the HMG box protein TCF7 as a key regulator in this self-renewal/differentiation switch, and it operates in the absence of canonical Wnt signaling. We found that TCF7 is the most downregulated transcription factor when CD34+ cells switch into CD34- cells using RNA-Seq. We subsequently identified the target genes bound by TCF7 using ChIP-Seq. We show that TCF7 binds to Runx1 (Aml1) promoter region, and RUNX1 and TCF7 co-regulate. Gene Set Enrichment Analysis suggests that TCF7 primarily acts as a positive regulator of genes preferentially expressed in CD34+ cells. Consistent with this possibility, knocking-down TCF7 represses many up-regulated genes in Lin-CD34+ cells. Finally a network of up-regulated transcription factors of CD34+ cells which defines the self-renewing state was constructed. These studies in EML cells demonstrate fundamental cell-intrinsic properties of the switch between self-renewal and differentiation, and yield valuable insights for manipulating HSCs and other differentiating systems. Examining the transcription factor binding targets of TCF7 and RUNX1.
Project description:We assess the concordance of histone H3 lysine 4 dimethylation (H3K4me2) and trimethylation (H3K4me3) on a genome-wide scale in erythroid development by analyzing pluripotent, multipotential and unipotent cell types. Although H3K4me2 and H3K4me3 are concordant at most genes, multipotential hematopoietic cells have a subset of genes that are differentially methylated (H3K4me2+/me3-). These genes are transcriptionally silent, highly enriched in lineage-specific hematopoietic genes, and uniquely susceptible to differentiation-induced H3K4 demethylation. Self-renewing embryonic stem cells, which restrict H3K4 methylation to genes that contain CpG islands (CGIs), lack H3K4me2+/me3- genes. These data reveal distinct epigenetic regulation of CGI and non-CGI genes during development and indicate an interactive relationship between DNA sequence and differential H3K4 methylation in lineage-specific differentiation. Keywords: comparison of different cell lines Multipotential and erythroid-differentiated hematopoietic cells were compared. Expression was measured using Affymetrix microarrays. H3K4me2 and H3K4me3 enrichment was assessed using Agilent tiling arrays.
Project description:Chronic lymphocytic leukemia (CLL), the most frequent adult leukemia in western countries, is a clonal accumulation of mature B-lymphocytes and its natural history is yet unclear. By using sequencing and cellular biology approaches on a cohort of CLL patient samples, we show here that acquired CLL mutations are observed in hematopoietic multipotent progenitor fractions in the majority of patients. These early CLL mutations include recurrent inactivating mutations in NFKBIE (10.7%) and missense mutations in BRAF (3.6%) and EGR2 (8.3%). Functional analyses demonstrated that BRAF-G469R affects lymphoid differentiation and transforms the T-cell lineage in vivo. In addition, the EGR2 recurrent mutations were associated with transcriptional activation of EGR2 target genes in patients and cell cycle abnormality in cellular model. Our findings indicate that CLL may develop from an initial infra-clinic, pre-leukemic phase affecting immature hematopoietic cells. The present study concerns gene expression of mouse EML cell line transfected by EGR2 wt gene and 2 mutated forms (E356K and H384N) and controls with the empty vector. Gene expression was performed in single color on Agilent 8x60K Mouse Genome array (design 028005).
Project description:Phenotypic cell-to-cell variability within clonal populations may be amanifestation of "gene expression noise", or it may reflect stablephenotypic variants. Such "non-genetic cell individuality" can arisefrom the slow fluctuations of protein levels in mammalian cells. Thesefluctuations produce persistent cell individuality, thereby rendering aclonal population heterogeneous. However, it remains unknown whetherthis heterogeneity may account for the stochasticity of cell fatedecisions in stem cells which depends on the kinetics that underliesheterogeneity. Here we show that in clonal populations of hematopoieticprogenitor cells, spontaneous "outlier" cells with extremely high or lowexpression levels of the stem cell marker Sca-1 reconstitute theparental distribution of Sca-1 but do so only after more than one week.This slow relaxation is described by a Gaussian-Mixture Model (GMM) thatincorporates noise-driven transitions between discrete subpopulations,suggesting hidden multi-stability within one cell type. Despiteclonality, the Sca-1 outliers had distinct transcriptomes. Although theunique gene expression profiles eventually reversed to that of themedian cells, they lasted long enough to confer a greatly differentproclivity for choosing either the erythroid or myeloid lineages. Preference in lineage choice was associated with elevated expression oflineage-specific transcription factors, such as > 200-fold increase inGATA1 among the erythroid-prone cells, or > 15-fold PU.1 expressionamong myeloid-prone cell. Thus, clonal heterogeneity of gene expressionlevel is not due to independent noise in the expression of individualgenes, but reflects metastable states of a slowly fluctuatingtranscriptome that is distinct in individual cells and may govern thereversible, stochastic priming of multipotent progenitor cells in cellfate decision. Keywords: murine EML cell line In the study presented here, the highest, middle, and lowest 15% Sca-1expressors from a clonal population of EML cells were used to acquireexpression profiles of a total of 46,628 unique genes using the IlluminaMouse-6 v1.1 microbead chips. Here, the Rank-Invariant normalized datais provided along with the individual raw data files.