Maturation stage–specific regulation of megakaryopoiesis by pointed-domain Ets proteins

Abstract Numerous megakaryocyte-specific genes contain signature Ets-binding sites in their regulatory regions. Fli-1 (friend leukemia integration 1), an Ets transcription factor, is required for the normal maturation of megakaryocytes and controls the expression of multiple megakaryocyte-specific genes. However, in Fli-1–/– mice, early megakaryopoiesis persists, and the expression of the early megakaryocyte-specific genes, αIIb and cMpl, is maintained, consistent with functional compensation by a related Ets factor(s). Here we identify the Ets protein GABPα (GA-binding protein α) as a regulator of early megakaryocyte-specific genes. Notably, GABPα preferentially occupies Ets elements of early megakaryocyte-specific genes in vitro and in vivo, whereas Fli-1 binds both early and late megakaryocyte-specific genes. Moreover, the ratio of GABPα/Fli-1 expression declines throughout megakaryocyte maturation. Consistent with this expression pattern, primary fetal liver–derived megakaryocytes from Fli-1–deficient murine embryos exhibit reduced expression of genes associated with late stages of maturation (glycoprotein [GP] Ibα, GPIX, and platelet factor 4 [PF4]), whereas GABPα-deficient megakaryocytes were mostly impaired in the expression of early megakaryocyte-specific genes (αIIb and cMpl). Finally, mechanistic experiments revealed that GABPα, like Fli-1, can impart transcriptional synergy between the hematopoietic transcription factor GATA-1 and its cofactor FOG-1 (friend of GATA-1). In concert, these data reveal disparate, but overlapping, functions of Ets transcription factors at distinct stages of megakaryocyte maturation.

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Maturation Stage-Specific Regulation of Megakaryocytic Genes by Pointed-Domain Ets Proteins.

Blood ◽

10.1182/blood.v106.11.1737.1737 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1737-1737

Author(s):

Liyan Pang ◽

Xun Wang ◽

Yuhuan Wang ◽

Gerd Blobel ◽

Mortimer Poncz

Keyword(s):

Gene Expression ◽

Fetal Liver ◽

Critical Role ◽

Regulatory Region ◽

Maturation Stage ◽

Specific Gene ◽

Mrna Levels ◽

Specific Regulation

Abstract The pointed-domain Ets transcription factor Fli-1 has a critical role during megakaryocyte-specific gene expression. Previously, we demonstrated that Fli-1 occupies the early megakaryocyte-specific gene αIIb in vivo. Moreover, our work suggested a mechanism for Fli-1 function by showing that Fli-1 facilitates GATA-1/FOG-1 dependent expression of the αIIb gene. However, studies by others with a targeted disruption of the Fli-1 gene in mice showed that while Fli-1 is essential for normal megakaryocyte maturation, αIIb mRNA levels were not significantly reduced in the resulting megakaryocytes, suggesting that a related Ets factor(s) might compensate for the loss of Fli-1. Here we show that the widely expressed pointed domain Ets protein GABPα specifically binds in vitro to Ets elements from two early megakaryocyte-specific genes, αIIb and c-mpl. Chromatin immunoprecipitation (ChIP) experiments using primary murine fetal liver-derived megakaryocytes reveal that GABPα associates with αIIb and c-mpl in vivo. Moreover, GABPα is capable of mediating GATA-1/FOG-1 synergy in the context of αIIb promoter constructs. These results suggest that GABPα contributes to megakaryocyte-restricted gene expression and is capable of at least partially compensating for the loss of Fli-1. However, loss of Fli-1 leads to a pronounced decrease in the expression of the late megakaryocyte-specific gene GPIX, indicating that compensation by GABPα is incomplete. Consistent with this observation, ChIP experiments fail to detect significant levels of GABPα at the regulatory region of GPIX while Fli-1 is readily detected there. Together, these results point to a model in which Fli-1 and GABPα serve overlapping, but distinct roles, during the development of megakaryocytes. GABPα may be important during early megakaryopoiesis, but Fli-1 exerting an essential role during late stages of maturation.

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The SCL Transcription Factor Supports Erythroid Cell Survival and Differentiation Downstream of the Erythropoietin Receptor.

Blood ◽

10.1182/blood.v104.11.818.818 ◽

2004 ◽

Vol 104 (11) ◽

pp. 818-818

Author(s):

Rachid Lahlil ◽

Richard Martin ◽

Peter D. Aplan ◽

C. Glenn Begley ◽

Jacqueline E. Damen ◽

...

Keyword(s):

Transcription Factor ◽

Cell Survival ◽

Cell Fate ◽

Genetic Interaction ◽

Fetal Liver ◽

Erythropoietin Receptor ◽

Erythroid Cell ◽

Loss Of Function

Abstract Erythroid cell development critically depends on the SCL/Tal1 transcription factor and on erythropoietin signalling. In the present study, we have taken several approaches to show that the two genes operate within the same pathway to consolidate the erythroid lineage. Signaling through the erythropoietin receptor (EpoR) upregulates SCL protein levels in a clonal cell line (TF-1) in vitro, and in murine fetal liver cells in vivo, when Epor−/− cells were compared to those of wild type littermates at E12.5. In addition, we provide functional evidence for a linear pathway from EpoR to SCL that regulates erythropoiesis. Interfering with SCL induction or SCL function prevents the anti-apoptotic effect of Epo in TF-1 cells and conversely, ectopic SCL expression is sufficient to substitute for Epo to transiently maintain cell survival. In vivo, SCL gain of function complements the cellular defects in Epor−/− embryos to support cell survival and maturation during primitive and definitive erythropoiesis, as assessed by cellular and histological analyses of Epor−/− SCLtg embryos. Moreover, several erythroid specific genes that are decreased in Epor−/− embryos are rescued by the SCL transgene including glycophorinA, bH1 and bmaj globin, providing molecular confirmation of the functional and genetic interaction between Epor and SCL. Conversely, erythropoiesis becomes deficient in compound Epor+/−SCL+/− heterozygote mice, indicating that the genetic interaction between EpoR and SCL is synthetic. Finally, using EpoR mutants that harbour well defined signalling deficiencies, combined with gain and loss of function approaches for specific kinases, we identify MAPK as the major signal transduction pathway downstream of EpoR that upregulates SCL function, necessary for erythroid cell survival and differentiation. Taken together, our observations are consistent with the view that cytokines can influence cell fate by altering the dosage of lineage transcriptional regulators.

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DNA Methylation Profile of Runx1 Regulatory Regions Is Correlated with Transition From Primitive to Definitive Hematopoietic Potential In Vitro and In Vivo

Blood ◽

10.1182/blood.v118.21.389.389 ◽

2011 ◽

Vol 118 (21) ◽

pp. 389-389

Author(s):

Beau Webber ◽

Michelina Iacovino ◽

Michael Kyba ◽

Bruce R. Blazar ◽

Jakub Tolar

Keyword(s):

Dna Methylation ◽

Stem Cells ◽

Transcription Factor ◽

Fetal Liver ◽

Methylation Status ◽

Differential Methylation ◽

Promoter Usage ◽

Highly Correlated

Abstract Abstract 389 Introduction: The Runt-related transcription factor Runx1 (AML1) is a central regulator of mammalian hematopoiesis and is required for the generation of hematopoietic stem cells (HSC) from hemogenic endothelium in the embryo. It has been shown that Runx1 is alternatively expressed from two promoters in a temporal fashion, and that their differential activities are influenced by a conserved intronic enhancer (+23) element. Intriguingly, promoter usage follows a pattern whereby the proximal (P2) initiates early in primitive hematopoiesis, while the distal (P1) becomes active later at the time of HSC emergence and is the predominant isoform expressed in fetal liver and adult HSC. While some transcription factor binding sites and cis-regulatory elements have been identified, an explanation for the alternative promoter usage remains elusive. We hypothesized that this regulation may be at the level of chromatin accessibility, and therefore investigated the DNA methylation status of Runx1 cis-elements. Methods/Results: We analyzed bisulfite-treated genomic DNA from E14.5 fibroblast (MEF), E8.5 yolk sac CD41+ (YS), E14.5 fetal liver Lin-Sca-1+CD48-CD150+ (FL), and adult marrow Lin-cKit+Sca-1+ (KLS); representing non-hematopoietic, primitive hematopoietic, and two stages of definitive HSC respectively. In addition, we also examined methylation in hematopoietic populations derived in vitro from murine embryonic stem cells (mESC). Initial exploratory analysis focused on classically defined CpG islands upstream of each promoter, however no significant differential methylation was observed within these regions. Subsequent analysis focused on CpGs near the transcription start site (TSS) and within the +23 enhancer. The P2 promoter was uninformative as it was unmethylated in all populations analyzed, whereas methylation within the +23 enhancer differentiated between hematopoietic and non-hematopoietic cell populations. At the P1 promoter, methylation status was remarkably correlated with primitive vs. definitive status. P1 was highly methylated in MEFs (77%), mESC embryoid body (EB) derived cKit+CD41+ (66%), and E8.5 YS CD41+ (58%); but significantly less methylated in vivo in FL HSC (8.1%) and adult KLS cells (18%). We are currently using this correlation of demethylation and definitive HSC potential to identify conditions that may drive definitive HSC generation from mESC-derived blood progenitors. Since overexpression of HoxB4 coupled with OP9 co-culture is the only confirmed method capable of producing definitive HSC from mESC, and HoxB4 has been shown to bind within the P1 promoter region of Runx1, we cultured HoxB4 or control EB-derived hematopoietic progenitors on OP9 stroma. We observed progressive demethylation in the HoxB4 arm: after 6 days of co-culture 47% vs. 71% in controls, and after 11 days 27% in the HoxB4 arm while the control population failed to proliferate past day 6. Isoform specific RT-PCR confirmed that HoxB4 overexpression resulted in Runx1 expression from the P1 promoter whereas the control vector did not. Within P1, we identify a single CpG that is most highly correlated with definitive HSC potential in vivo, and most significantly demethylated upon HoxB4 overexpression in vitro. Conclusions: These data indicate that differential methylation occurs at Runx1 regulatory regions during hematopoietic development in vitro and in vivo. The +23 enhancer is demethylated in cells with hematopoietic potential, whereas demethylation of the Runx1 P1 promoter is highly correlated with definitive HSPC populations and is promoted in vitro by HoxB4. These data are the first to identify a role for DNA methylation in the regulation of alternative promoter usage at the Runx1 locus, and may serve as a novel biomarker of HSC potential during embryonic development. Disclosures: No relevant conflicts of interest to declare.

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The N terminus of Ascl1 underlies differing proneural activity of mouse and Xenopus Ascl1 proteins

Wellcome Open Research ◽

10.12688/wellcomeopenres.14842.1 ◽

2018 ◽

Vol 3 ◽

pp. 125 ◽

Cited By ~ 3

Author(s):

Laura J.A. Hardwick ◽

Anna Philpott

Keyword(s):

Transcription Factor ◽

Bhlh Transcription Factor ◽

Protein Accumulation ◽

Primary Neuron ◽

Helix Loop Helix ◽

N Terminus ◽

Specific Regulation ◽

Species Specific

The proneural basic-helix-loop-helix (bHLH) transcription factor Ascl1 is a master regulator of neurogenesis in both central and peripheral nervous systems in vivo, and is a central driver of neuronal reprogramming in vitro. Over the last three decades, assaying primary neuron formation in Xenopus embryos in response to transcription factor overexpression has contributed to our understanding of the roles and regulation of proneural proteins like Ascl1, with homologues from different species usually exhibiting similar functional effects. Here we demonstrate that the mouse Ascl1 protein is twice as active as the Xenopus protein in inducing neural-β-tubulin expression in Xenopus embryos, despite there being little difference in protein accumulation or ability to undergo phosphorylation, two properties known to influence Ascl1 function. This superior activity of the mouse compared to the Xenopus protein is dependent on the presence of the non-conserved N terminal region of the protein, and indicates species-specific regulation that may necessitate care when interpreting results in cross-species experiments.

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Mechanisms of Tumor Suppression by the Hematopoietic Transcription Factor PU.1.

Blood ◽

10.1182/blood.v120.21.2432.2432 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2432-2432

Author(s):

Mark D McKenzie ◽

Luisa Cimmino ◽

Yifang Hu ◽

Ladina Di Rago ◽

Sandra Mifsud ◽

...

Keyword(s):

Transcription Factor ◽

Transgenic Mice ◽

Myeloid Leukemia ◽

Target Genes ◽

Fetal Liver ◽

Liver Cells ◽

Myeloid Lineage ◽

Fetal Liver Cells

Abstract Abstract 2432 Introduction Acute myeloid leukemia (AML) is a genetically and morphologically heterogeneous disease characterized by the accumulation of immature myeloid lineage cells in the bone marrow and blood. It results from genetic alterations that cause increased self-renewal of myeloid progenitors, accompanied by a block in their normal differentiation programs. Studies in mice and humans have shown that loss of expression of PU.1, a master transcription factor that is critical for lymphoid and myeloid lineage development, is a recurrent feature of AML1. Restoring the PU.1 differentiation program in AML is an attractive therapeutic strategy, but remains elusive due to a poor understanding of PU.1 target genes and tumor suppressive mechanisms. In a novel approach to understanding PU.1 function, we have used in vivo RNA interference to inducibly inhibit and restore PU.1 expression in normal hematopoietic cells and leukemias. Results PU.1 knockdown promotes leukemia in mice We identified several short hairpin RNAs that can effectively knockdown PU.1 (Fig 1A). We infected primary fetal liver cells with the most effective LMP-shPU.1 retroviruses and performed in vitro and in vivo assays to assess the effect of PU.1 knockdown (Fig 1B). We found that PU.1 knockdown drives 1) an increased frequency of blast colony-forming cells and self-generation of granulocytic progenitors in vitro (Fig 1C) and 2) a GFP+ myeloid leukemia after several months characterized by accumulation of cKit+Gr1+Mac1+ cells (Fig 1D, E). These findings verify that shRNA-mediated PU.1 knockdown can effectively disable its tumor suppressive functions. Inducible restoration of PU.1 in leukemia in vivo To identify transcriptional targets of PU.1 in vivo, we utilized a recently generated reversible RNAi strategy that allows acute restoration of endogenous PU.1 expression upon Dox treatment in leukemias driven by PU.1 knockdown2. This TRMPV vector strategy allows tet-regulated co-expression of an shRNA and the fluorescent marker dsRed, with stable expression of GFP to mark infected cells. We transduced fetal liver cells derived from Vav-tTA transgenic mice with TRMPV-shPU.1 to drive reversible PU.1 knockdown across the hematopoietic system of reconstituted recipient mice. In contrast to the myeloid leukemia generated earlier using LMP-shPU.1, these mice developed pre-B cell (CD19+CD25+) leukemia with a latency of several months. To acutely restore endogenous PU.1 expression in leukemia, primary tumor cells were transplanted into several recipient mice to generate a cohort for analysis of Dox responses (Figure 2A). We found that dsRed intensity decreased incrementally upon Dox treatment of leukemic transplant recipient mice allowing FACS sorting of leukemia cells from triplicate untreated mice (dsRedhigh, minimal PU.1 expression) or after three days of Dox treatment (dsRedmid, partially restored PU.1 expression). We identified gene expression changes associated with PU.1 restoration using RNA sequencing (RNA-seq). Development of a transgenic mouse allowing inducible PU.1 knockdown in vivo To further investigate PU.1 target genes in vivo, we have recently generated TRE-GFP-shPU.1 transgenic mice allowing inducible knockdown and restoration of PU.1 in adult mice. To test this strain we crossed it to CAGs-rtTA3 mice and treated bitransgenic mice with Dox. Western blot analysis of GFP+ Gr1+Mac1+ sorted myeloid cells showed effective PU.1 knockdown in vivo. We are currently using these mice to identify PU.1 regulated genes in normal myeloblasts in vivo. Conclusions These studies have identified several new candidate PU.1-regulated genes. Further experiments may shed light on whether there is a common novel tumor suppressive mechanism for PU.1 in myeloid and lymphoid leukemias driven by loss of PU.1. Disclosures: No relevant conflicts of interest to declare.

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Bacterial Nucleoid-Associated Protein Uncouples Transcription Levels from Transcription Timing

mBio ◽

10.1128/mbio.01485-14 ◽

2014 ◽

Vol 5 (5) ◽

Cited By ~ 24

Author(s):

Igor Zwir ◽

Won-Sik Yeo ◽

Dongwoo Shin ◽

Tammy Latifi ◽

Henry Huang ◽

...

Keyword(s):

Transcription Factor ◽

Target Genes ◽

Transcriptional Regulator ◽

Mrna Levels ◽

Content Type ◽

Expression Of Genes ◽

Serovar Typhimurium ◽

Expression Behavior

ABSTRACTThe histone-like nucleoid-structuring (H-NS) protein binds to horizontally acquired genes in the bacteriumSalmonella entericaserovar Typhimurium, silencing their expression. We now report that overcoming the silencing effects of H-NS imposes a delay in the expression of genes activated by the transcriptional regulator PhoP. We determine that PhoP-activated genes ancestral toSalmonellaare expressed before those acquired horizontally. This expression timing reflects thein vivooccupancy of the corresponding promoters by the PhoP protein. These results are surprising because some of these horizontally acquired genes reached higher mRNA levels than ancestral genes expressed earlier and were transcribed from promoters harboring PhoP-binding sites with higherin vitroaffinity for the PhoP protein. Our findings challenge the often-made assumption that for genes coregulated by a given transcription factor, early genes are transcribed to higher mRNA levels than those transcribed at later times. Moreover, they provide a singular example of how gene ancestry can impact expression timing.IMPORTANCEWe report that gene ancestry dictates the expression behavior of genes under the direct control of theSalmonellatranscriptional regulator PhoP. That is, ancestral genes are transcribed before horizontally acquired genes. This reflects both the need to overcome silencing by the H-NS protein of the latter genes and the architecture of the corresponding promoters. Unexpectedly, transcription levels do not reflect transcription timing. Our results illustrate how a bacterium can exhibit an elaborate temporal expression behavior among genes coregulated by a transcription factor even though the products encoded by the target genes do not participate in a morphological or developmental pathway.

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Functional interaction of the v-Rel and c-Rel oncoproteins with the TATA-binding protein and association with transcription factor IIB

Molecular and Cellular Biology ◽

10.1128/mcb.13.11.6733-6741.1993 ◽

1993 ◽

Vol 13 (11) ◽

pp. 6733-6741

Author(s):

X Xu ◽

C Prorock ◽

H Ishikawa ◽

E Maldonado ◽

Y Ito ◽

...

Keyword(s):

Transcription Factor ◽

Binding Protein ◽

Tata Binding Protein ◽

Basal Transcription ◽

Transcription Factor Iib ◽

Expression Of Genes ◽

C Terminus ◽

Domain Mapping

Rel family proteins regulate the expression of genes linked to kappa B-binding motifs. Little is known, however, of the mechanism by which they enhance transcription. We have investigated the ability of the v-Rel and c-Rel oncoproteins to interact with components of the basal transcription machinery. Here we report that both the acidic transcription activation domain mapping to the unique C terminus of chicken c-Rel and the F9 cell-specific activation region common to both v-Rel and c-Rel interact with the TATA-binding protein (TBP) and transcription factor IIB (TFIIB) in vitro and in vivo. We also demonstrate that TPB interaction with Rel activation regions leads to synergistic activation of transcription of a kappa B-linked reporter gene. Combined with the observation that the mouse c-Rel and human RelA proteins also interact with TBP and TFIIB in vitro, these results suggest that association with basal transcription factors is important for the transcriptional activities of Rel family proteins.

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Differentiation-Dependent Interactions between RUNX-1 and FLI-1 during Megakaryocyte Development

Molecular and Cellular Biology ◽

10.1128/mcb.00090-09 ◽

2009 ◽

Vol 29 (15) ◽

pp. 4103-4115 ◽

Cited By ~ 58

Author(s):

Hui Huang ◽

Ming Yu ◽

Thomas E. Akie ◽

Tyler B. Moran ◽

Andrew J. Woo ◽

...

Keyword(s):

Transcription Factor ◽

Protein Interactions ◽

Transcriptional Activation ◽

Fetal Liver ◽

Gel Filtration ◽

Cell Types ◽

Protein Protein Interactions ◽

Megakaryocyte Differentiation

ABSTRACT The transcription factor RUNX-1 plays a key role in megakaryocyte differentiation and is mutated in cases of myelodysplastic syndrome and leukemia. In this study, we purified RUNX-1-containing multiprotein complexes from phorbol ester-induced L8057 murine megakaryoblastic cells and identified the ets transcription factor FLI-1 as a novel in vivo-associated factor. The interaction occurs via direct protein-protein interactions and results in synergistic transcriptional activation of the c-mpl promoter. Interestingly, the interaction fails to occur in uninduced cells. Gel filtration chromatography confirms the differentiation-dependent binding and shows that it correlates with the assembly of a complex also containing the key megakaryocyte transcription factors GATA-1 and Friend of GATA-1 (FOG-1). Phosphorylation analysis of FLI-1 with uninduced versus induced L8057 cells suggests the loss of phosphorylation at serine 10 in the induced state. Substitution of Ser10 with the phosphorylation mimic aspartic acid selectively impairs RUNX-1 binding, abrogates transcriptional synergy with RUNX-1, and dominantly inhibits primary fetal liver megakaryocyte differentiation in vitro. Conversely, substitution with alanine, which blocks phosphorylation, augments differentiation of primary megakaryocytes. We propose that dephosphorylation of FLI-1 is a key event in the transcriptional regulation of megakaryocyte maturation. These findings have implications for other cell types where interactions between runx and ets family proteins occur.

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HNF1alpha is involved in tissue-specific regulation of CFTR gene expression

Biochemical Journal ◽

10.1042/bj20031157 ◽

2004 ◽

Vol 378 (3) ◽

pp. 909-918 ◽

Cited By ~ 44

Author(s):

Nathalie MOUCHEL ◽

Sytse A. HENSTRA ◽

Victoria A. McCARTHY ◽

Sarah H. WILLIAMS ◽

Marios PHYLACTIDES ◽

...

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Tissue Specificity ◽

Regulatory Elements ◽

Upstream Region ◽

Mrna Levels ◽

Tissue Specific ◽

Specific Regulation

The CFTR (cystic fibrosis transmembrane conductance regulator) gene shows a complex pattern of expression with tissue-specific and temporal regulation. However, the genetic elements and transcription factors that control CFTR expression are largely unidentified. The CFTR promoter does not confer tissue specificity on gene expression, suggesting that there are regulatory elements outside the upstream region. Analysis of potential regulatory elements defined as DNase 1-hypersensitive sites within introns of the gene revealed multiple predicted binding sites for the HNF1α (hepatocyte nuclear factor 1α) transcription factor. HNF1α, which is expressed in many of the same epithelial cell types as CFTR and shows similar differentiation-dependent changes in gene expression, bound to these sites in vitro. Overexpression of heterologous HNF1α augmented CFTR transcription in vivo. In contrast, antisense inhibition of HNF1α transcription decreased the CFTR mRNA levels. Hnf1α knockout mice showed lower levels of CFTR mRNA in their small intestine in comparison with wild-type mice. This is the first report of a transcription factor, which confers tissue specificity on the expression of this important disease-associated gene.

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