Large-Scale Integration of Gene Profiling Identifies TCF7L2/TCF4 as the Most Frequently Dysregulated Wnt Signaling Component In AML

Abstract Abstract 2480 Acute myeloid leukemia (AML) represents one of the most genetically heterogeneous malignancies; however, some processes are commonly dysregulated. One of the most frequently dysregulated processes in AML is Wnt signaling. In solid cancers, aberrant Wnt signaling has been shown to promote cancer by increasing nuclear accumulation of β-catenin and with consequent activation of target genes. In AML, overexpression of β-catenin is also common; in addition however, patient studies and genetic models indicate that other components of the Wnt pathway are also commonly dysregulated and may mediate transcriptional changes independently of β-catenin. The aim of this study was to identify aberrantly regulated Wnt components and target genes in AML by interactome analysis of the AML Affymetrix GeneChip® 3` expression microarray datasets; a network building algorithm used to understand relationships between genes. Analysis and interpretation of microarray data is still both biologically and computationally challenging. To address this, we performed batch adjustment to the large scale AML dataset by merging gene expression profile (GEP) data derived from different database sources (including different array platforms). GEPs data were generated from our AML patients enrolled in two different AML NCRI-MRC UK clinical trials using two different Affymetrix platforms, HG-U133A (n=216) and HG-U133Plus2.0 (n=139). GEPs from normal CD34+ bone marrow samples were downloaded from ArrayExpress (n=26). In order to compare AML vs. normal haematopoietic GEP, all data were merged into a single dataset. Individual. CEL files were imported into Partek® Genomics Suite™ and GC-RMA normalization was applied. Linear contrasts, mixed model analysis of variance with false discovery rate correction (P<0.05) and threshold analysis (>1.5 or <1.5 fold-change) were applied to the adjusted data followed by gene enrichment analysis using MetaCore™ (GeneGo Inc). Batch adjustment was performed using Distance Weight Discrimination (DWD) method to the merged GEPs. Prior to further inferential and gene ontology testing, the DWD merged datasets showed significant reduction in the source of data bias with GEP clustered according to their biological variation rather than technical variation. As a result, we present a final list of 58 significant changes in the expression of Wnt related genes in AML. Enrichment by protein function analysis highlighted 8 Wnt transcription factors to be dysregulated (TCF7L2/TCF4, MYC, NANOG, WT1, RUNX2, p300, TCF7, SMAD2), along with 5 receptors (CD44, FZD3, FZD4, FZD5, LDLR), 3 types of phosphatases (B56G, PR61-β, PPP2R5A) and other categories of Wnt related objects (n=33). Consistent findings were seen with previously established Wnt-associated genes specific to AML (CD44, WT1, MYC) showing that data sources from DWD adjustment was effective. We sought to evaluate the significant biological and functional relationships within the genes in the final dataset by transcription factor target modeling using MetaCore™ Interactome tools. Direct network interaction uncovered TCF7L2/TCF4 as the most significantly upregulated Wnt transcription factor with concurrent high expression of its downstream Wnt responsive genes CD44, AXIN1, ID2 that were also present in the final list. Importantly, β-catenin is unlikely to contribute to this transcriptional activation due to the fact that our data showed increased transcription of β-catenin degradation complexes (or negative regulation of Wnt signalling). Specifically, RUVBL1, that directly increases β-catenin activity was significantly downregulated, whereas the other significantly overexpressed upstream genes (APC, CSNK1E, AXIN1, WT1) are known to have inhibitory effect on β-catenin-mediated transcription. In summary, by using multiple GEP data from a large AML cohort in conjunction with robust statistical adjustments, we have identified TCF7L2/TCF4 mediated transcription as the most significant Wnt-regulated process to be altered in AML compared with normal blasts. We also predict that transcription of TCF7L2/TCF4 regulated genes is likely to be independent of β-catenin, supporting observations in genetic models which indicate that β-catenin (and γ-catenin) are redundant for normal haematopoiesis and are not required for TCF-mediated transcription. Disclosures: No relevant conflicts of interest to declare.

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Transcriptional Silencing of the Wnt-Antagonist Dickkopf-1 (DKK1) by Promoter Methylation Unleashes Aberrant Wnt Signaling In Advanced Multiple Myeloma

Blood ◽

10.1182/blood.v116.21.1919.1919 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1919-1919

Author(s):

Kinga A Kocemba ◽

Richard W Groen ◽

Harmen van Andel ◽

Karene Mahtouk ◽

Marie Jose Kersten ◽

...

Keyword(s):

Multiple Myeloma ◽

Wnt Signaling ◽

Cell Lines ◽

Wnt Pathway ◽

Conflicts Of Interest ◽

Cpg Island ◽

Nuclear Accumulation ◽

Aberrant Methylation ◽

Dkk1 Expression ◽

Osteolytic Bone Disease

Abstract Abstract 1919 Aberrant activation of the Wnt/β-catenin pathway is implicated in driving the formation of various human cancers. Recent studies indicate that the Wnt pathway plays at least two distinct roles in the pathogenesis of multiple myeloma (MM): i) Aberrant, presumably autocrine, activation of canonical Wnt signaling in MM cells promotes tumor proliferation and metastasis; ii) Overexpression of the Wnt inhibitor Dickkopf1 (DKK1), contributes to osteolytic bone disease by inhibiting osteoblast differentiation. Since DKK1 itself is a target of TCF/β-catenin mediated transcription, these findings suggests the presence of a negative feedback loop in MM, in which DKK1 acts as a potential tumor suppressor. In line with this hypothesis, we show here that DKK1 expression is lost in most MM cell lines and in a subset of patients with advanced MM. This loss is correlated with activation of the Wnt pathway, as demonstrated by increased nuclear accumulation of β-catenin. Analysis of the DKK1 promoter revealed CpG island methylation in several MM cell lines as well as in MM cells from patients with advanced MM. Moreover, demethylation of the DKK1 promoter restores DKK1 expression, which results in inhibition of β-catenin/TCF-mediated gene transcription in MM lines. Taken together, our data identify aberrant methylation of the DKK1 promoter as a cause of DKK1 silencing in advanced stage MM, which may play an important role in the progression of MM by unleashing Wnt signaling. Disclosures: No relevant conflicts of interest to declare.

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C/ebpα and Pu.1 Are Members of a Critical Regulatory Network Required for Hoxa9-Mediated Immortalization

Blood ◽

10.1182/blood.v120.21.650.650 ◽

2012 ◽

Vol 120 (21) ◽

pp. 650-650

Author(s):

Cailin Collins ◽

Jingya Wang ◽

Joel Bronstein ◽

Jay L. Hess

Keyword(s):

Transcription Factor ◽

Binding Sites ◽

Target Genes ◽

De Novo ◽

Conflicts Of Interest ◽

Epigenetic Modifications ◽

Conditional Knockout ◽

Motif Analysis ◽

Myeloid Leukemias

Abstract Abstract 650 HOXA9 is a homeodomain-containing transcription factor that plays important roles in both development and hematopoiesis. Deregulation of HOXA9 occurs in a variety of acute lymphoid and myeloid leukemias and plays a key role in their pathogenesis. More than 50% of acute myeloid leukemia (AML) cases show up-regulation of HOXA9, which correlates strongly with poor prognosis. Nearly all cases of AML with mixed lineage leukemia (MLL) translocations have increased HOXA9 expression, as well as cases with mutation of the nucleophosmin gene NPM1, overexpression of CDX2, and fusions of NUP98. Despite the crucial role that HOXA9 plays in development, hematopoiesis and leukemia, its transcriptional targets and mechanisms of action are poorly understood. Previously we identified Hoxa9 and Meis1 binding sites in myeloblastic cells, profiled their epigenetic modifications, and identified the target genes regulated by Hoxa9. Hoxa9 and Meis1 co-bind at hundreds of promoter distal, highly evolutionarily conserved sites showing high levels of histone H3K4 monomethylation and CBP/p300 binding characteristic of enhancers. Hoxa9 association at these sites correlates strongly with increases in histone H3K27 acetylation and activation of downstream target genes, including many proleukemic gene loci. De novo motif analysis of Hoxa9 binding sites shows a marked enrichment of motifs for the transcription factors in the C/EBP and ETS families, and C/ebpα and the ETS transcription factor Pu.1 were found to cobind at Hoxa9-regulated enhancers. Both C/ebpα and Pu.1 are known to play critical roles in the establishment of functional enhancers during normal myeloid development and are mutated or otherwise deregulated in various myeloid leukemias. To determine the importance of co-association of Hoxa9, C/ebpα and Pu.1 at myeloid enhancers, we generated cell lines from C/ebpα and Pu.1 conditional knockout mice (kindly provided by Dr. Daniel Tenen, Harvard University) by immortalization with Hoxa9 and Meis1. In addition we transformed bone marrow with a tamoxifen-regulated form of Hoxa9. Strikingly, loss of C/ebpα or Pu.1, or inactivation of Hoxa9, blocks proliferation and leads to myeloid differentiation. ChIP experiments show that both C/ebpα and Pu.1 remain bound to Hoxa9 binding sites in the absence of Hoxa9. After the loss of Pu.1, both Hoxa9 and C/ebpα dissociate from Hoxa9 binding sites with a corresponding decrease in target gene expression. In contrast, loss of C/ebpα does not lead to an immediate decrease in either Hoxa9 or Pu.1 binding, suggesting that C/ebpα may be playing a regulatory as opposed to a scaffolding role at enhancers. Current work focuses on performing ChIP-seq analysis to assess how C/ebpα and Pu.1 affect Hoxa9 and Meis1 binding and epigenetic modifications genome-wide, and in vivo leukemogenesis assays to confirm the requirement of both Pu.1 and C/ebpα in the establishment and maintenance of leukemias with high levels of Hoxa9. Collectively, our findings implicate C/ebpα and Pu.1 as members of a critical transcription factor network required for Hoxa9-mediated transcriptional regulation in leukemia. Disclosures: No relevant conflicts of interest to declare.

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Gfi1 Upregulates c-Myc Expression and Promotes c-Myc-Driven Cell Proliferation

Blood ◽

10.1182/blood-2019-124833 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3769-3769

Author(s):

Yangyang Zhang ◽

Fan Dong

Keyword(s):

Cell Proliferation ◽

Transcription Factor ◽

Transcriptional Repression ◽

Cell Cycle Progression ◽

Target Genes ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Protein Levels ◽

Enhanced Expression ◽

Ubiquitin Proteasome

Gfi1 is a zinc-finger transcriptional repressor that plays an important role in hematopoiesis. When aberrantly activated, Gfi1 may function as a weak oncoprotein in the lymphoid system, but collaborate strongly with c-Myc in lymphomagenesis. c-Myc is a transcription factor that is frequently activated in human cancers including leukemia and lymphoma mainly due to its overexpression as a result of gene amplifications and chromosomal translocations. c-Myc overexpression may also result from stabilization of c-Myc protein, which is highly unstable and rapidly degraded through the ubiquitin-proteasome pathway. The mechanism by which Gfi1 collaborates with c-Myc in lymphomagenesis is incompletely understood. c-Myc activates gene expression by forming a heterodimeric complex with the partner protein Max, but may also repress target genes through interaction with transcription factor Miz-1. We previously showed that Gfi1 indirectly interacts with c-Myc through Miz-1 and collaborates with c-Myc to repress CDK inhibitors p21Cip1 and p15Ink4B. In this study, we show that Gfi1 augmented the level of c-Myc protein transiently expressed in Hela cells and the levels of MycER fusion protein stably expressed in the mouse pro-B Ba/F3 and myeloid 32D cells. The C-terminal ZF domains of Gfi1, but not its transcriptional repression and DNA binding activities, were required for c-Myc upregulation. Notably, although Miz-1 has been shown to stabilize c-Myc protein, the expression of c-Myc V394D mutant, which is defective in Miz-1 interaction, was still upregulated by Gfi1, suggesting that Gfi1-mediated c-Myc upregulation was independent of Miz-1 interaction. We further show that Gfi1 overexpression led to reduced polyubiquitination and increased stability of c-Myc protein. Interestingly, the levels of endogenous c-Myc mRNA and protein were augmented upon induction of Gfi1 expression in Ba/F3 and Burkitt lymphoma Ramos cells transduced with the doxycycline-inducible Gfi1 lentiviral construct, but reduced in Gfi1-knocked down human leukemic HL60 and U937 cells. Additionally, targeted deletion of Gfi1 resulted in reduced c-Myc expression in mouse lineage negative bone marrow cells, which was associated with a decline in the expression of c-Myc-activated target genes. The oncogenic potential of Myc derives from its ability to stimulate cell proliferation. Our results demonstrate that inducible expression of Gfi1 in Ba/F3 cells expressing MycER promoted Myc-driven cell cycle progression and proliferation. Thus, in addition to its role in c-Myc-mediated transcriptional repression, Gfi1 upregulates c-Myc expression at both mRNA and protein levels, leading to enhanced expression of c-Myc-activated genes and augmented cell proliferation driven by c-Myc. Together, these data may reveal a novel mechanism by which Gfi1 collaborates with c-Myc in lymphomagenesis. Disclosures No relevant conflicts of interest to declare.

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Hepatocyte nuclear factor-1β regulates Wnt signaling through genome-wide competition with β-catenin/lymphoid enhancer binding factor

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1909452116 ◽

2019 ◽

Vol 116 (48) ◽

pp. 24133-24142 ◽

Cited By ~ 3

Author(s):

Siu Chiu Chan ◽

Ying Zhang ◽

Marco Pontoglio ◽

Peter Igarashi

Keyword(s):

Transcription Factor ◽

Wnt Signaling ◽

Target Genes ◽

Cystic Kidney ◽

Nuclear Factor ◽

Hepatocyte Nuclear Factor ◽

Wild Type ◽

Genome Wide ◽

Binding Factor ◽

Mutant Cells

Hepatocyte nuclear factor-1β (HNF-1β) is a tissue-specific transcription factor that is essential for normal kidney development and renal tubular function. Mutations of HNF-1β produce cystic kidney disease, a phenotype associated with deregulation of canonical (β-catenin–dependent) Wnt signaling. Here, we show that ablation of HNF-1β in mIMCD3 renal epithelial cells produces hyperresponsiveness to Wnt ligands and increases expression of Wnt target genes, including Axin2, Ccdc80, and Rnf43. Levels of β-catenin and expression of Wnt target genes are also increased in HNF-1β mutant mouse kidneys. Genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) in wild-type and mutant cells showed that ablation of HNF-1β increases by 6-fold the number of sites on chromatin that are occupied by β-catenin. Remarkably, 50% of the sites that are occupied by β-catenin in HNF-1β mutant cells colocalize with HNF-1β–occupied sites in wild-type cells, indicating widespread reciprocal binding. We found that the Wnt target genes Ccdc80 and Rnf43 contain a composite DNA element comprising a β-catenin/lymphoid enhancer binding factor (LEF) site overlapping with an HNF-1β half-site. HNF-1β and β-catenin/LEF compete for binding to this element, and thereby HNF-1β inhibits β-catenin–dependent transcription. Collectively, these studies reveal a mechanism whereby a transcription factor constrains canonical Wnt signaling through direct inhibition of β-catenin/LEF chromatin binding.

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Multiple transcription factors co-regulate the Mycobacterium tuberculosis adaptation response to vitamin C

BMC Genomics ◽

10.1186/s12864-019-6190-3 ◽

2019 ◽

Vol 20 (1) ◽

Cited By ~ 1

Author(s):

Malobi Nandi ◽

Kriti Sikri ◽

Neha Chaudhary ◽

Shekhar Chintamani Mande ◽

Ravi Datta Sharma ◽

...

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Mycobacterium Tuberculosis ◽

Vitamin C ◽

Large Scale ◽

Target Genes ◽

Transcriptional Regulatory Network ◽

Transcriptional Regulators ◽

Transcriptional Regulatory ◽

Modulation Of Gene Expression

Abstract Background Latent tuberculosis infection is attributed in part to the existence of Mycobacterium tuberculosis in a persistent non-replicating dormant state that is associated with tolerance to host defence mechanisms and antibiotics. We have recently reported that vitamin C treatment of M. tuberculosis triggers the rapid development of bacterial dormancy. Temporal genome-wide transcriptome analysis has revealed that vitamin C-induced dormancy is associated with a large-scale modulation of gene expression in M. tuberculosis. Results An updated transcriptional regulatory network of M.tuberculosis (Mtb-TRN) consisting of 178 regulators and 3432 target genes was constructed. The temporal transcriptome data generated in response to vitamin C was overlaid on the Mtb-TRN (vitamin C Mtb-TRN) to derive insights into the transcriptional regulatory features in vitamin C-adapted bacteria. Statistical analysis using Fisher’s exact test predicted that 56 regulators play a central role in modulating genes which are involved in growth, respiration, metabolism and repair functions. Rv0348, DevR, MprA and RegX3 participate in a core temporal regulatory response during 0.25 h to 8 h of vitamin C treatment. Temporal network analysis further revealed Rv0348 to be the most prominent hub regulator with maximum interactions in the vitamin C Mtb-TRN. Experimental analysis revealed that Rv0348 and DevR proteins interact with each other, and this interaction results in an enhanced binding of DevR to its target promoter. These findings, together with the enhanced expression of devR and Rv0348 transcriptional regulators, indicate a second-level regulation of target genes through transcription factor- transcription factor interactions. Conclusions Temporal regulatory analysis of the vitamin C Mtb-TRN revealed that there is involvement of multiple regulators during bacterial adaptation to dormancy. Our findings suggest that Rv0348 is a prominent hub regulator in the vitamin C model and large-scale modulation of gene expression is achieved through interactions of Rv0348 with other transcriptional regulators.

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The transcription factor Schnurri plays a dual role in mediating Dpp signaling during embryogenesis

Development ◽

10.1242/dev.128.9.1657 ◽

2001 ◽

Vol 128 (9) ◽

pp. 1657-1670 ◽

Cited By ~ 1

Author(s):

J. Torres-Vazquez ◽

S. Park ◽

R. Warrior ◽

K. Arora

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Cell Fate ◽

Target Genes ◽

Gene Activation ◽

Nuclear Accumulation ◽

Embryonic Patterning ◽

Specific Analysis ◽

Positive Input ◽

Affect Gene Expression

Decapentaplegic (Dpp), a homolog of vertebrate bone morphogenic protein 2/4, is crucial for embryonic patterning and cell fate specification in Drosophila. Dpp signaling triggers nuclear accumulation of the Smads Mad and Medea, which affect gene expression through two distinct mechanisms: direct activation of target genes and relief of repression by the nuclear protein Brinker (Brk). The zinc-finger transcription factor Schnurri (Shn) has been implicated as a co-factor for Mad, based on its DNA-binding ability and evidence of signaling dependent interactions between the two proteins. A key question is whether Shn contributes to both repression of brk as well as to activation of target genes. We find that during embryogenesis, brk expression is derepressed in shn mutants. However, while Mad is essential for Dpp-mediated repression of brk, the requirement for shn is stage specific. Analysis of brk; shn double mutants reveals that upregulation of brk does not account for all aspects of the shn mutant phenotype. Several Dpp target genes are expressed at intermediate levels in double mutant embryos, demonstrating that shn also provides a brk-independent positive input to gene activation. We find that Shn-mediated relief of brk repression establishes broad domains of gene activation, while the brk-independent input from Shn is crucial for defining the precise limits and levels of Dpp target gene expression in the embryo.

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The Hbo1-Brd1/Brpf2 HAT Complex Is Required for Erythropoiesis In Fetal Liver

Blood ◽

10.1182/blood.v116.21.3872.3872 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3872-3872

Author(s):

Yuta Mishima ◽

Satoru Miyagi ◽

Atsunori Saraya ◽

Masamitsu Negishi ◽

Mitsuhiro Endoh ◽

...

Keyword(s):

Transcription Factor ◽

Target Genes ◽

Conflicts Of Interest ◽

Fetal Liver ◽

Flow Cytometric Analysis ◽

K562 Cells ◽

Erythroid Differentiation ◽

Pwwp Domain ◽

Transcription Factor Genes ◽

Chip Analysis

Abstract Abstract 3872 Bromodomain-containing protein 1 (Brd1, initially designated as BR140-LIKE; BRL) contains a bromodomain, two plant homology domain (PHD) zinc fingers, and a proline-tryptophan-tryptophan-proline (PWWP) domain, three types of modules characteristic of chromatin regulators. Recently, BRD1 appeared to belong to the BRPF family which includes BRPF1, BRD1/BRPF2, and BRPF3. Among them, BRPF1 is known to be a subunit of the MOZ H3 histone acetyltransferase (HAT) complex. BRD1 has been proposed to be additional subunit of the MOZ H3 HAT complex on the analogy of BRPF1. However, its molecular function remains elusive. To elucidate the biological functions of BRD1, we generated Brd1-null mice and found that they die in utero. Brd1-/- embryos were alive and recovered at nearly the expected Mendelian ratio at 12.5 days postcoitum (dpc) but died by 15.5 dpc. Brd1-/- embryos at 12.5 dpc were pale and the cell number of fetal livers, in which fetal hematopoiesis occurs, was decreased to about 20% of the control. Cytological analysis revealed that Brd1-/- fetal livers had profoundly fewer erythroblasts at maturation stages beyond proerythroblasts compared to wild-type fetal livers. Flow cytometric analysis of Brd1-/- fetal livers revealed a significant accumulation of CD71+Ter119- proerythroblasts and a reduction in CD71+Ter119+ and CD71-Ter119+ maturating erythroblasts. A drastic increase in AnnexinV+ apoptotic cells was detected in the CD71+Ter119+ and CD71-Ter119- cell fractions in Brd1-/- fetal livers. These findings suggested that severe anemia caused by compromised differentiation and/or survival of erythroblasts accounts for embryonic lethality of Brd1-/- embryos. To understand the mechanism underlying defective erythropoiesis in Brd1-null embryos, we performed biochemical analyses and found that Brd1 bridges the HAT, HBO1 but not MOZ, and its activator protein, ING4, to form an enzymatically active HAT complex. Forced expression of Brd1 promoted erythroid differentiation of K562 cells, while Brpf1, which preferentially binds to MOZ, had no significant effect. Correspondingly, depletion of Hbo1 by Hbo1 knockdown perturbed erythroid differentiation of mouse fetal liver progenitors. Of note, the level of global acetylation of histone H3 at lysine 14 (H3K14) was specifically decreased in Brd1-deficient erythroblasts. These results collectively implied that acetylation of H3K14 catalyzed by the Hbo1-Brd1 complex has a crucial role in fetal liver erythropoiesis. To identify the downstream targets for the HBO1-BRD1 complex, we performed the ChIP-on-chip analysis in K562 cells and found that BRD1 and HBO1 largely co-localize on the genome, especially on the promoters of erythroid transcription factor genes. ChIP analysis revealed that acetylation of H3K14 at the promoters of erythroid transcription factor genes, including Gata1, Gata2, Tal1, Stat5a, and ETO2, were profoundly diminished in the Brd1-deficient erythroblasts. Among these target genes, we focused on Gata1, which plays a central role in erythropoiesis, and carried out complementation experiments with Gata1 using a Gata1 retrovirus. Exogenous Gata1, but not Bcl-xL, efficiently improved proliferative capacity and survival of Brd1-deficient erythroid progenitors and also restored, at least partially, their impaired differentiation. These results clearly showed that the Hbo1-Brd1 complex is required for the acetylation of H3K14 at the promoters of erythroid transcription factor genes, thereby is crucial for erythropoiesis in fetal liver. Disclosures: No relevant conflicts of interest to declare.

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GATA2 Transcription Factor Mediates Myeloid Expansion Coupled with Lymphoid Block by Transcriptional Regulation of Key Target Genes.

Blood ◽

10.1182/blood.v120.21.2317.2317 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2317-2317

Author(s):

Satish Kumar Nandakumar ◽

Geoffrey A. Neale ◽

Derek A. Persons

Keyword(s):

Transcription Factor ◽

Transcriptional Regulation ◽

Cell Lines ◽

Liquid Culture ◽

Insertional Mutagenesis ◽

Target Genes ◽

Conflicts Of Interest ◽

Myeloid Cell ◽

Control Group ◽

Mouse Bone Marrow

Abstract Abstract 2317 Functions of the transcription factor GATA2 in the hematopoietic system were difficult to explore due to the embryonic lethal phenotype on deletion (Tsai et al., Nature 1994) and HSC quiescence on overexpression (Persons et al., Blood 1999). To overcome this problem we developed a Tamoxifen inducible system by fusing the human GATA2 cDNA to the ligand binding domain of the estrogen receptor (GATA2-ERT). Previously we found that when competitive transplant experiments were performed with mouse bone marrow cells, GATA2-ERT transduced cells greatly outcompeted untransduced myeloid cells while at the same time not contributing to the lymphoid lineage. However, no leukemia was observed. Since these effects were observed in the absence of Tamoxifen, we speculated that GATA2-ERT protein leaked into the nucleus to provide low level tonic activity (known to occur in other ERT systems). Immunoblot analysis of BM cells transduced with the GATA2-ERT vector confirmed the presence of the protein in both nuclear and cytoplasmic fractions. In addition, GATA2-ERT transduced cells also out-competed mock transduced cells in liquid culture and could be serially replated in colony forming assays. GATA2-ERT and control GFP transduced cells were diluted with mock cells to achieve around 25% GFP+ cells. After 9 days in culture the percentage of GFP + cells in the GATA2-ERT group was 91%±6 compared to GFP group which remained at 38%±14. GATA2-ERT transduced cells could be cultured indefinitely while after 7 weeks the control GFP transduced cells differentiated to mast cells. These GATA2-ERT cell lines were dependent only on IL-3 and expressed myeloid cell surface markers (53%±1 Gr+, 87%±4 Mac1+ cells n=3). DNA binding mutants of GATA2-ERT completely abolished both serial colony replating ability and growth advantage in liquid culture. This suggested that GATA2-ERT mediates these effects through transcriptional regulation of target genes. To identify the targets of GATA2 responsible for myeloid expansion, gene expression profiling was done on three independently generated GATA2-ERT cell lines and compared to an immortalized myeloid cell line (due to insertional mutagenesis) which does not express GATA2. Genes involved in myeloid neoplasms like Nmyc, HoxA9 and Bcl11a were significantly elevated (HoxA9 –77-fold, Nmyc 60-fold, Bcl11a 36-fold) in the GATA2-ERT lines. To test if GATA2 mediates myeloid expansion through Nmyc, we knocked down Nmyc in GATA2-ERT cells and tested for growth defects. GATA2-ERT cells were transduced with lentiviral vectors harboring shRNA targeting GATA2-ERT (73%±10.2), Nmyc (77%±9.5) and scrambled (75%±7) and cultured for 18 days. As expected, transduced cell numbers dropped to 5%±1 for GATA2-ERT group, confirming GATA2 dependence, while transduced cells decreased to 40%±8 for the Nmyc group with the control group unchanged. This suggests that Nmyc could be one of the key targets of GATA2 responsible for myeloid expansion. Current studies are aimed at determining whether GATA2 directly regulates the Nmyc promoter. Disclosures: No relevant conflicts of interest to declare.

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Gene Expressional Profile of Neonatal Neutrophils in Response to Bacterial Lipopolysaccharide (LPS) and Possible Mechanism of Transcription Factor NR4A2 on the Immune Signal Cascade

Blood ◽

10.1182/blood.v126.23.995.995 ◽

2015 ◽

Vol 126 (23) ◽

pp. 995-995

Author(s):

Kathy Yuen Yee Chan ◽

Ka Ian Tam ◽

Kam Tong Leung ◽

Tak Yeung Leung ◽

Karen Li ◽

...

Keyword(s):

Transcription Factor ◽

Cord Blood ◽

Target Genes ◽

Conflicts Of Interest ◽

Developmental Regulation ◽

Pyrrolidine Dithiocarbamate ◽

Male Adult ◽

Gram Negative ◽

Innate Defense ◽

Signal Cascade

Abstract Gram-negative bacterial infection is a serious condition in neonates which could lead to septicemic shock, disseminated intravascular coagulation and death. LPS, the cell wall component of Gram-negative bacteria is a potent stimulator of the host immune response system, mediated by binding to toll-like receptor (TLR)-4 and CD14 receptors. Neutrophils are the first line of innate defense which possess antimicrobial and acute inflammatory activities. Neonatal neutrophils, though incompletely characterized, are suggested to be functionally immature and compromised in their bactericidal capacity compared with adults. We performed a comparative genome-wide expression array analysis on purified (>95%) human cord blood (term delivery) neutrophils upon challenge by LPS for 4 hours at 100 ng/mL. With the criteria of 2-fold differences and statistical significance (P <0.01), 1236 transcripts were increased and 1633 transcripts were decreased in LPS-treated neutrophils (n=3). A panel of 20 target genes was selected by their magnitude of regulation, novelty and relevant immune functions and validated by qPCR. Eleven of these genes could be linked upstream or downstream of the novel transcription factor, nuclear receptor subfamily-4, group-A, member-2 (NR4A2) by pathway analysis (Metacore). We thus proposed a regulatory network mediated by binding of LPS to TLR4/CD14 receptors, leading to NR4A2 upregulation, thereby promoting a cascade of downstream effector genes associated with antibacterial and/or inflammatory activities in neutrophils. To validate the upstream signal cascade of LPS-induced regulation of NR4A2, neonatal neutrophils were pretreated with specific TLR-4, p38MAPK or NF-κB inhibitors, and stimulated with LPS, followed by quantification of target gene expression by qPCR (n=6 in each group). Our results showed that LPS-induced NR4A2 expression was significantly decreased (all P <0.05) after pretreatment with TLR-4 antibody (0.47 fold), p38MAPK inhibitor SB203580 (0.27 fold), and NF-κB inhibitors BAY-117082 (0.14 fold) and pyrrolidine dithiocarbamate (0.07 fold). These results suggested that NR4A2 could be upregulated by LPS-receptor-mediated p38MAPK and NF-κB signals. To compare the capacity and developmental regulation of LPS-induced responses of NR4A2-associated signals, purified cord blood neutrophils from preterm (gestational age 33-36 weeks) and term (37-40 weeks) neonates, and peripheral blood from healthy male adult (age 25-30 years old) were evaluated (n=10 in each group). LPS-induced expression of NR4A2 and associated molecular signals (EGR1, PPARG, NF-κB, ATOH8, G0S2, GPR84, IL-8, PTGS2, TNF-α, CAMK2G and GCH1) were measured by qPCR. Our result showed that expressions of these target genes were significantly up- or downregulated in LPS-activated neutrophils of neonates and adults. However, at the basal level, expressions of EGR1, GOS2, IL-8 and PTSG2 were significantly lower (P< 0.05) in preterm or term neonatal neutrophils compare with those in adults, showing that there might be compromised levels in neonates at the unstimulated status. Upon stimulation with LPS, expressions of GOS2 and IL-8 were significantly lower (P< 0.05) in preterm or term neonates than in adult, indicating that there could be developmental regulation of NR4A2 downstream genes in neutrophils secondary to LPS stimulations. Correlation analysis showed that the expressions of EGR1 and PTGS2 were positively correlated with NR4A2 in LPS-induced neutrophils from both term (EGR1: R=0.805, P< 0.05; PTGS2: R=0.912, P< 0.05) and preterm neonates (EGR1: R=0.636, P< 0.05; PTGS2: R=0.830, P< 0.05), but not in adult peripheral neutrophils. These results indicated that there could be developmental differences in the regulation of the NR4A2 immune cascade involving apoptosis (EGR1, GOS2)/inflammatory cytokine (IL-8) and prostaglandin synthesis (PTGS2) between adult and neonatal neutrophils at basal and/or LPS-activation states. In summary, we reported the expressional profile and deregulated target genes in neonatal neutrophils upon stimulation by LPS and identified the downstream NR4A2 immune cascade that could be regulated by LPS, p38MAPK and NF-κB signals, as well as the developmental status. Disclosures No relevant conflicts of interest to declare.

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Fli1 Expression Level Affects Megakaryopoiesis and Thrombopoiesis: More Is Not Always a Bad Thing

Blood ◽

10.1182/blood.v128.22.3876.3876 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3876-3876

Author(s):

Karen K Vo ◽

Danuta Jadwiga Jarocha ◽

Randolph B Lyde ◽

Spencer K. Sullivan ◽

Deborah French ◽

...

Keyword(s):

Transcription Factor ◽

Genome Editing ◽

Platelet Activation ◽

Transgene Expression ◽

Conflicts Of Interest ◽

Function Analysis ◽

Ipsc Line ◽

Protein Levels ◽

Platelet Disorder

Abstract Friend leukemia integration 1 (FLI1) is a critical transcription factor responsible for terminal megakaryocyte differentiation. This transcription factor is amongst the genes missing in the inherited disorder Jacobsen syndrome, resulting from a hemizygous deletion on chromosome 11q. The deletion causes dysmegakaryopoiesis and macrothrombocytopenia termed Paris Trousseau syndrome (PTSx). Also, FLI1 mutations in its DNA-binding domain region results in thrombocytopenia in affected patients. We examined induced pluripotent stem cell- (iPSC) derived megakaryocytes (iMegs) to determine if the platelet disorder observed in PTSx could be replicated and if varied levels of Fli1 expression affected megakaryopoiesis and thrombopoiesis. Beginning with a normal control (WT) iPSC line, genome editing was performed to generate three lines with: 1) one copy of FLI1 disrupted (FLI1+/-), 2) hemizygous transgene expression of Fli1 in the adeno-associated virus site 1 (AAVS1) safe harbor locus using a megakaryocyte-specific GP1balpha promoter (WT-overexpressing line, WT-OE), and 3) homozygous transgene expression in AAVS1 (WT-OE2). Additionally, we established an iPSC line from a PTSx patient and edited this line for a similar hemizygous transgene expression of FLI1 (PTSx-OE). Data described here are summarized in the tables below. We confirm our genome editing strategies by examining mRNA and protein levels of iMegs and found WT-OE and WT-OE2 iMegs have ~3X higher mRNA and ~7X higher protein levels than WT iMegs. FLI1+/- and PTSx iMegs both have lower mRNA and ~0.5X the protein levels of WT iMegs, while PTSx-OE iMegs had comparable levels of mRNA and protein as WT-OE iMegs. Megacult colony assays showed WT-OE and WT-OE2 yielded more CFU-Megs than WT HPCs (p²0.01, p²0.05, respectively). PTSx and FLI1+/- had much less CFU-Megs compared to PTSx-OE and WT (p²0.0001). After growth in liquid culture, WT-OE and -OE2 lines had an increase in iMeg numbers (p=0.29, p²0.01) while FLI1+/- and PTSx iMeg numbers were 43% and 10% that of the WT control (p²0.01, p²0.001). PTSx-OE iMeg numbers were comparable to WT. Surface marker CD41 and CD42b levels were increased compared to WT iMegs in the WT-OE and -OE2 iMegs (p=0.25, p²0.05) and less on FLI1+/- and PTSx iMegs (p²0.01, p²0.05). PTSx-OE iMegs were normal compared to WT, but p²0.05 vs. PTSx. Infused CD41+CD42b+ iMegs into NSG mice showed a trend toward same yield of platelets in the OE lines and lower yield in FLI1+/- and PTSx lines compared to WT. However, when calculations were made from HPCs rather than iMegs infused, the FLI1+/- and PTSx iMegs generated significantly less number of released platelets (p²0.05, p²0.01). The half-life of WT-OE and -OE2 released platelets were increased at 4 and 10 hours compared to WT, whereas FLI1+/- and PTSx released platelets have lower half-lives of 2 hours and 0.5 hours, respectively. These decreased platelet half-lives are due to the majority of platelets being defective and cleared at a higher rate. This was corrected to WT in the PTSx-OE-released platelets. Both in vitro iMegs and in vivo released platelets within the murine blood were assessed for function. Analysis of activation by thrombin was performed via FACS for PAC-1 binding and cell surface P-selectin levels, which are both indicators of platelet activation. Initial studies show no difference between WT and the WT-OE and WT-OE2 lines, but FLI1+/- platelet activation was slightly impaired. In summary, we show that studies of iMegs with decreased Fli1 levels replicate many of the clinical features previously described: less iMegs, lower CD41 and CD42b density with less platelets released while having shorter half-lives. On the other hand, increased Fli1 levels resulted in higher number of iMegs with more surface antigen density and released platelets with increased half-lives. Based on overexpression studies of other transcription factors during megakaryopoiesis, such as GATA1, the expectation would have been that the excess of Fli1 in the OE lines would have also resulted in defects in megakaryopoiesis and thrombopoiesis, but instead improvements were seen. The basis for this difference when overexpressing different transcriptional factors needs further analysis. That high Fli1 levels enhance iMeg yield with maintained numbers and function of released platelets may be of value for generating platelets for clinical use from in vitro grown Megs. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

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