Transcription Factors Regulating Early Hematopoietic Development and Lineage Commitment

1998 ◽  
pp. 41-54 ◽  
Author(s):  
Stuart H. Orkin
Keyword(s):  
Transcription Factors ◽  
Lineage Commitment ◽  
Hematopoietic Development

Development of Lymphohematopoietic Progenitors during Human Embryonic Stem (hES) Cell Differentiation on OP9 Stromal Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2789-2789
Author(s):  
Igor I. Slukvin ◽  
Maxim A. Vodyanik ◽  
Jack A. Bork ◽  
James A. Thomson
Keyword(s):  
Endothelial Cells ◽  
Transcription Factors ◽  
Cell Differentiation ◽  
Stromal Cells ◽  
Embryonic Stem ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Development ◽  
Cd34 Cells ◽  
Transient Population ◽  
Hes Cells

Abstract hES cells provide an unique opportunity to study the earliest stages of hematopoietic commitment which are not easily accessible in the human embryo. To model early hematopoietic development, we cultured H1 and H9 hES cell lines on OP9 stromal cells without the addition of cytokines. On day 2 of co-culture, hES cells up-regulated brachyury expression and began to form mesodermal-like colonies. A transient population of blast colony-forming cells (CFCs) with the potential to differentiate into blood and endothelial cells was detected on days 3–6 of co-culture. CD34+ cells first appeared on day 3–4 of co-culture, which was coincident with induction of the transcription factors GATA-1, GATA-2 and SCL. CD43+ and CD41a+ cells along with CFCs emerged 2 days later within CD34+ population; 3–4 days before the appearance of CD45+ cells. We were able to obtain up to 20% of CD34+ cells from hES/OP9 co-culture and isolate up to 107 CD34+ cells with more than 95% purity from a similar number of initially plated hES cells after 8–9 days of culture. The hES cell-derived CD34+ cells were highly enriched in CFCs, displayed CD90+CD117+CD164+CD38- phenotype of primitive hematopoietic progenitors, and contained ALDHhigh cells as well cells with verapamil-sensitive ability to efflux rhodamine 123. Isolated CD34+ cells differentiated into lymphoid (NK cells) as well as myeloid (neutrophils and macrophages) lineages when cultured on MS-5 stromal cells in the presence of SCF, Flt3-L, IL7 and IL3. These data indicate that hES cell/OP9 co-culture reproduces the major events that are observed during embryonal hematopoietic development, including the formation of lympho-myeloid progenitors. We employed OP9 system for identification of the phenotype of early hematopoietic progenitors in humans and to directly differentiate hES cells into different blood lineages.

Molecular Background of BCP-ALL Cases with an Early Switch to Monocytic Lineage

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3562-3562
Author(s):  
Karel Fišer ◽  
Lucie Slámová ◽  
Alena Dobiášová ◽  
Júlia Starková ◽  
Eva Froňková ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Transcription Factors ◽  
B Cell ◽  
Differentially Expressed ◽  
Lineage Commitment ◽  
Altered Expression ◽  
B Lineage ◽  
And Control ◽  
Monocytic Lineage

Abstract We identified a subset of BCP-ALL with switch towards the monocytic lineage within the first month of treatment (swALL)[Slámová et al Leukemia 2014]. During the switch cells gradually lose CD19 and CD34 expression and acquire CD33 and CD14 positivity. We proved clonal relatedness of switched monocytic blasts with the diagnostic leukemic cells based on identical Ig-TCR rearrangements. SwALL cases are not associated with MLL or BCR/ABL1 aberrancies and lack any known genetic markers of lineage ambiguity (detected by FISH or MLPA). We analyzed transcriptomes of swALL samples at diagnosis (n=4) and at d8 (n=4) where the immunophenotypic switching was already apparent as well as control BCP-ALL (n=4). RNA was isolated form either FACS sorted cells or whole BM when blasts constituted >80% of cells. For RNA-Seq we used Illumina HiSeq 2000 paired-end or single end sequencing. Raw sequencing data were analyzed using adapted protocol from Anders at al [Anders et al Nature Protocols 2013] and custom scripts. For methylome analysis we used Enhanced Reduced Representation Bisulfite Sequencing (ERRBS)[Akalin et al PLoS Genetics 2012]. ERRBS quantitatively measures DNA methylation at ~3M CpGs genome-wide. Samples from swALL at diagnosis (n=7) and at d8 (n=4) and control BCP-ALL (n=4) were processed. Analysis was performed according to [Akalin et al Genome Biology 2012] and followed with custom analysis in R statistical language. Comparison (generalized exact binomial test) of transcriptomes of B-lineage blasts from diagnosis between swALLs and control BCP-ALLs revealed a number of differentially expressed genes. Among 300 most significantly differentially expressed were KLF4, CEBPD, CLEC12A and CLEC12B (upregulated in swALL) and ANXA5, VPREB1, CD9 and IGHG3 (downregulated in swALL). Hierarchical clustering separated not only swALL and control BCP-ALL, but also swALL cells before and during the monocytic switch. Changes in gene expression during lineage switch included downregulation of ITGA6, Id2, EBF1, CD19, CD34, FLT3, MYB, CD79a, BCR, PAX5, GATA3 and TCF3 genes and upregulation of S100A10, AIF1, CD14, CD33, LGALS1, RNF130 and MNDA. When comparing all three cell types (swALL B cell and monocytic blasts and control BCP-ALL blasts) we concentrated on 1) immunophenotype switch markers and 2) lineage related transcription factors (TF): 1) Both markers typical for B cell blasts (CD19, CD34) decreased during the switch. However while CD19 was expressed in swALL at diagnosis at same levels as in control BCP-ALL, CD34 was overexpressed in swALL compared to BCP-ALL at diagnosis. Both monocytic markers (CD33, CD14) increased their expression during the switch. CD14 showed no difference between swALL and control BCP-ALL at diagnosis. However CD33 was interestingly upregulated in swALL already at diagnosis and continued to rise during the switch. SwALL had therefore deregulated expression of lineage commitment markers already at diagnosis favoring stemness marker CD34 and myeloid marker CD33. 2) B lineage commitment related TFs (EBF1, TCF3, PAX5) were expressed in B lineage blasts in both swALL and control BCP-ALL. However they were all downregulated during the switch. On the other hand myeloid lineage related transcription factor CEBPA is overexpressed in diagnostic B lineage blasts in swALL compared to control BCP-ALL cases. Similarly CEBPD is overexpressed in swALL and its expression further rises during the switch. Other hematopoietic TFs upregulated in swALL cases include KLF4, NANOG and GATA3. To confirm some of the epigenetic markers of swALL cases (demethylation of CEBPA promoter) and to widen epigenetic screening we used ERRBS. While some of the upregulated genes had expectedly hypomethylated promoters in swALL (CEBPA, GATA3) other genes (TCF3, PAX5) had demethylated promoters in all cases. While the whole DNA methylation picture is still a challenge to draw both omics method could clearly separate swALL cases from control BCP-ALL using principal component analysis. In summary we show that immunophenotypic shift is associated with gene expression changes of surface markers, lineage specific transcription factors and other genes. Some of the genes have altered expression already at diagnosis. Expression of some key lineage genes is differentially regulated by DNA methylation. Supported by: GAUK 914613, GAČR P301/10/1877, UNCE 204012, IGA NT13462-4 Disclosures No relevant conflicts of interest to declare.

scholarly journals Precise developmental regulation of Ets family transcription factors during specification and commitment to the T cell lineage

Development ◽  
1999 ◽  
Vol 126 (14) ◽  
pp. 3131-3148 ◽  
Author(s):  
M.K. Anderson ◽  
G. Hernandez-Hoyos ◽  
R.A. Diamond ◽  
E.V. Rothenberg
Keyword(s):  
Transcription Factors ◽  
T Cell ◽  
B Cell ◽  
Developmental Stages ◽  
Developmental Regulation ◽  
Hematopoietic Cells ◽  
Cell Lineage ◽  
Lineage Commitment ◽  
Lineage Specification ◽  
Ets Family

Ets family transcription factors control the expression of a large number of genes in hematopoietic cells. Here we show strikingly precise differential expression of a subset of these genes marking critical, early stages of mouse lymphocyte cell-type specification. Initially, the Ets family member factor Erg was identified during an arrayed cDNA library screen for genes encoding transcription factors expressed specifically during T cell lineage commitment. Multiparameter fluorescence-activated cell sorting for over a dozen cell surface markers was used to isolate 18 distinct primary-cell populations representing discrete T cell and B cell developmental stages, pluripotent lymphoid precursors, immature NK-like cells and myeloid hematopoietic cells. These populations were monitored for mRNA expression of the Erg, Ets-1, Ets-2, Fli-1, Tel, Elf-1, GABPalpha, PU.1 and Spi-B genes. The earliest stages in T cell differentiation show particularly dynamic Ets family gene regulation, with sharp transitions in expression correlating with specification and commitment events. Ets, Spi-B and PU.1 are expressed in these stages but not by later T-lineage cells. Erg is induced during T-lineage specification and then silenced permanently, after commitment, at the beta-selection checkpoint. Spi-B is transiently upregulated during commitment and then silenced at the same stage as Erg. T-lineage commitment itself is marked by repression of PU.1, a factor that regulates B-cell and myeloid genes. These results show that the set of Ets factors mobilized during T-lineage specification and commitment is different from the set that maintains T cell gene expression during thymocyte repertoire selection and in all classes of mature T cells.

scholarly journals Stage-specific action of Runx1 and GATA3 controls silencing of PU.1 expression in mouse pro–T cells

2021 ◽  
Vol 218 (8) ◽  
Author(s):  
Hiroyuki Hosokawa ◽  
Maria Koizumi ◽  
Kaori Masuhara ◽  
Maile Romero-Wolf ◽  
Tomoaki Tanaka ◽  
...  
Keyword(s):  
T Cells ◽  
Transcription Factor ◽  
Transcription Factors ◽  
T Cell ◽  
Cell Line ◽  
Functional Site ◽  
Lineage Commitment ◽  
Factor Binding ◽  
Ets Family ◽  
Intron 2

PU.1 (encoded by Spi1), an ETS-family transcription factor with many hematopoietic roles, is highly expressed in the earliest intrathymic T cell progenitors but must be down-regulated during T lineage commitment. The transcription factors Runx1 and GATA3 have been implicated in this Spi1 repression, but the basis of the timing was unknown. We show that increasing Runx1 and/or GATA3 down-regulates Spi1 expression in pro–T cells, while deletion of these factors after Spi1 down-regulation reactivates its expression. Leveraging the stage specificities of repression and transcription factor binding revealed an unconventional but functional site in Spi1 intron 2. Acute Cas9-mediated deletion or disruption of the Runx and GATA motifs in this element reactivates silenced Spi1 expression in a pro–T cell line, substantially more than disruption of other candidate elements, and counteracts the repression of Spi1 in primary pro–T cells during commitment. Thus, Runx1 and GATA3 work stage specifically through an intronic silencing element in mouse Spi1 to control strength and maintenance of Spi1 repression during T lineage commitment.

scholarly journals SHP-2 and canonical PD-1: SHP-2 axis regulate myeloid cell differentiation, anti-tumor responses and innate immune memory

2022 ◽  
Author(s):  
Vassiliki Boussiotis ◽  
Anthos Christofides ◽  
Xanthi-Lida Katopodi ◽  
Carol Cao ◽  
Halil-Ibrahim Aksoylar ◽  
...  
Keyword(s):  
Transcription Factors ◽  
T Cell ◽  
Tumor Growth ◽  
Tumor Immunity ◽  
Antigen Processing ◽  
Innate Immune ◽  
Lineage Commitment ◽  
Immune Memory ◽  
Myeloid Differentiation ◽  
Gm Csf

Abstract PD-1 checkpoint inhibitor induces T cell inactivation by recruiting SHP-2. However, T cell-specific SHP-2-deficient mice do not have improved anti-tumor immunity. We generated mice with conditional targeting of the Ptpn11 gene (encoding for Shp-2) in T cells (Shp2f/fLckCre) or myeloid cells (Shp2f/fLysMCre), and found that Shp2f/fLysMCre mice had diminished tumor growth. As determined by RNA-seq, this was paralleled by the presence of inflammatory neutrophils and tumor-associated macrophages (TAMs) with molecular signatures of enhanced differentiation, phagocytosis and antigen-processing and presentation. SHP-2 deficient TAMs also had increased monocyte and dendritic cell (DC) specification transcription factors, chemokine and cytokine production, and expression of immunostimulatory molecules that promote T cell recruitment and activation. Monocytes from tumor-bearing Shp2f/fLysMCre mice suppressed tumor growth after transfer to naïve recipients indicating development of innate immune memory. In bone marrow myelocytes, GM-CSF, induced PD-1 expression, phosphorylation and interaction with SHP-2, the Src family kinase Lyn, and GM-CSF receptor beta chain, indicating that the PD-1:SHP-2 axis targets a key pathway of myelocyte differentiation. In contrast, SHP-2 deletion or antibody-mediated blockade of the PD-1:PD-L1 pathway enhanced phosphorylation of the transcription factors HOXA10 and IRF8 that regulate myeloid differentiation and monocytic/moDC lineage commitment, respectively. Thus, SHP-2 and the PD-1:SHP-2 axis pose a signaling restrain to myeloid differentiation and monocyte lineage commitment resulting in a myeloid landscape that suppresses anti-tumor immunity.

PU.1 and GATA-1, but Not SCL Elicit an Increase in General Transcription Associated with the Lineage Restriction of Multipotent Progenitors.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4212-4212
Author(s):  
Michael Cross ◽  
Zoe McIvor ◽  
Dietger W. Niederwieser
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Transient Expression ◽  
Transcriptional Activity ◽  
Erythroid Differentiation ◽  
Lineage Commitment ◽  
Self Renewal ◽  
Multipotent Progenitors ◽  
Target Promoters ◽  
Target Promoter

Abstract The lineage commitment of multipotent progenitor cells is coordinated by regulatory interactions between transcription factor programs associated with different lineage fates. Accordingly, mutations which interfere with these programs can block differentiation and contribute to leukemogenesis. A thorough understanding of lineage commitment processes should therefore identify targets and strategies for differentiation therapy. We have compared the effects of transient over-expression of the hematopoietic transcription factors PU.1, GATA-1 and SCL on the response of a multipotent hematopoietic progenitor cell line (FDCPmix) to conditions supporting either self-renewal, myeloid differentiation or erythroid differentiation. Methods: Pure populations of FDCPmix cells productively transfected with transcription factor cDNA/IRES/GFP expression vectors were isolated by FACS sorting and entered directly into liquid media containing either IL-3 (for self-renewal); GM-CSF + G-CSF (for myeloid differentiation) or erythropoietin + hemin (for erythroid differentiation). Semi-solid medium containing IL-3 was used to assess the maintenance of that sub-population of cells which can undergo self-renewal in isolation, and which are assumed to represent the earliest progenitors in an FDCPmix culture. Transcription factor function was confirmed by co-transfection with reporter genes driven by specific target promoters. Results: The yield of FDCPmix cells from self-renewal medium was markedly enhanced by the transient expression of SCL but reduced by either PU.1 or GATA-1. These differences in yield reflect alterations in survival and/or proliferation over the two day period of transgene expression immediately following transfection. As expected, the transient expression of PU.1 severely reduced the potential of multipotent progenitors to respond to erythroid growth factors, while GATA-1 similarly reduced the response to myeloid growth factors, consistent with an antagonistic relationship between myeloid and erythroid transcription factors during lineage restriction. However, while all three transcription factors demonstrated the appropriate activity on their respective target promoters, PU.1 and GATA-1 also increased general transcriptional activity. Transient expression of GATA-1 actually raised the activity of the cfms (PU.1 target) promoter, while PU.1 activated the serpin 2A (GATA-1 target) promoter. Of the transcription factors tested, only PU.1 consistently reduced the frequency of colony forming cells, suggesting that the earliest multipotent progenitors in an FDCPmix population can be recruited to commitment by PU.1 but not by GATA-1. Conclusion: These results are consistent with a multi-step process of lineage commitment in which general transcriptional activity in multipotent progenitors is maintained at a low level, base state. An increase in general transcriptional activity would then be required as an early event in commitment, activating the transcription factor networks subsequently responsible for coordinating lineage restriction.

Transcriptional Regulation in Normal and Malignant Hematopoiesis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. SCI-28-SCI-28
Author(s):  
Koichi Akashi
Keyword(s):  
Transcription Factors ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Histone Variant ◽  
Lineage Commitment ◽  
Regulation Of Transcription ◽  
Hematopoietic Stem ◽  
Genome Wide ◽  
Regulated Expression ◽  
Transcription Factor Expression

Abstract SCI-28 Lineage commitment should involve selective and temporally-regulated expression of essential genes. In multi- or oligo-potent progenitors, the expression of oligo-lineage-affiliated genes is primed: oligo-lineage genes are co-expressed prior to the commitment at the single cell level, and once lineage fate is decided, genes of irrelevant lineages are immediately downregulated. For example, single common myeloid progenitors (CMP) co-express both granulocyte/monocyte-affiliated and megakaryocyte/erythroid-affiliated genes. The priming of these lineage-restricted genes could be dependent also upon the priming of lineage-specific transcription factors. Consistent with this hypothesis, a population with potent CMP activity that co-express both PU.1 (myeloid) and GATA-1 (erythroid) transcription factors was newly identified by using mice having PU.1 and GATA-1 reporters. In downstream of such “priming” stage, the order of expression as well as the level of expression of multiple transcription factors plays a critical role in reading-out specific lineages. The precise regulation of transcription factors should be critical to maintain hematopoietic homeostasis, and the deregulation of transcription factor expression could induce leukemic transformation. To understand the regulation machinery upstream of transcription factors, we are currently attempting to model an epigenetic landscape in hematopoietic development. Genome-wide analysis of histone positioning revealed that a histone variant marks hematopoietic transcription factors and other lineage-related genes prior to commitment, and therefore the variant can predict the actively-transcribed region in a later stage of hematopoiesis. For example, in hematopoietic stem cells, the histone marking was observed broadly in genes-related to myelo-erythroid and lymphoid genes, while in committed progenitors, it was restricted to specific lineages such as myeloid, erythroid and/or lymphoid genes. These results suggest that the genome marking by a histone variant should be a primary event for initiating lineage commitment. Disclosures: No relevant conflicts of interest to declare.

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