scholarly journals Opposing transcriptional mechanisms regulateToxoplasmadevelopment

2016 ◽  
Author(s):  
Dong-Pyo Hong ◽  
Joshua B. Radke ◽  
Michael W. White
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Cyst Formation ◽  
Tissue Cyst ◽  
Luciferase Reporter ◽  
Alkaline Stress ◽  
Developmental Pathway ◽  
Reciprocal Effect ◽  
Gene Target ◽  
Term Survival

ABSTRACTTheToxoplasmabiology that underlies human chronic infection is developmental conversion of the acute tachyzoite stage into the latent bradyzoite stage. We investigated the role of two alkaline-stress induced ApiAP2 transcription factors, AP2IV-3 and AP2IX-9, in bradyzoite development. These factors were expressed in two overlapping waves during bradyzoite development with AP2IX-9 increasing expression earlier than AP2IV-3, which peaked as AP2IX-9 expression was declining. Disruption of the AP2IX-9 gene enhanced, while deletion of AP2IV-3 gene decreased tissue cyst formation demonstrating these factors have opposite functions in bradyzoite development. Conversely, conditional overexpression of FKBP-modified of AP2IX-9 or AP2IV-3 with the small molecule Shield 1 had a reciprocal effect on tissue cyst formation confirming the conclusions of the knockout experiments. The AP2IX-9 repressor and AP2IV-3 activator tissue cyst phenotypes were borne out in gene expression studies that determined many of the same bradyzoite genes were regulated in an opposite manner by these transcription factors. A common gene target was the canonical bradyzoite marker, BAG1, and mechanistic experiments determined that like AP2IX-9, AP2IV-3 regulates a BAG1 promoter-luciferase reporter and specific binds the BAG1 promoter in parasite chromatin. Altogether, these results suggest the AP2IX-9 transcriptional repressor and AP2IV-3 transcriptional activator likely compete to control bradyzoite gene expression, which may permitToxoplasmato better adapt to different tissue environments and select a suitable host cell for long term survival of the dormant tissue cyst.IMPORTANCEToxoplasmainfections are life-long due to the development of the bradyzoite tissue cyst, which is effectively invisible to the immune system. Despite the important clinical consequences of this developmental pathway, the molecular basis of the switch mechanisms that control formation of the tissue cyst is still poorly understood. Significant changes in gene expression are associated with tissue cyst development and ApiAP2 transcription factors are an important mechanism regulating this developmental transcriptome. However, the molecular composition of these ApiAP2 mechanisms are not well defined and the operating principles of ApiAP2 mechanisms are poorly understood. Here we establish that competing ApiAP2 transcriptional mechanisms operate to regulate this clinically important developmental pathway.

scholarly journals Opposing Transcriptional Mechanisms Regulate Toxoplasma Development

mSphere ◽  
2017 ◽  
Vol 2 (1) ◽  
Author(s):  
Dong-Pyo Hong ◽  
Joshua B. Radke ◽  
Michael W. White
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Immune System ◽  
Molecular Basis ◽  
Cyst Formation ◽  
Molecular Composition ◽  
Tissue Cyst ◽  
Developmental Pathway ◽  
Clinical Consequences ◽  
Cyst Development

ABSTRACT Toxoplasma infections are lifelong because of the development of the bradyzoite tissue cyst, which is effectively invisible to the immune system. Despite the important clinical consequences of this developmental pathway, the molecular basis of the switch mechanisms that control tissue cyst formation is still poorly understood. Significant changes in gene expression are associated with tissue cyst development, and ApiAP2 transcription factors are an important mechanism regulating this developmental transcriptome. However, the molecular composition of these ApiAP2 complexes and the operating principles of ApiAP2 mechanisms are not well defined. Here we establish that competing ApiAP2 transcriptional mechanisms operate to regulate this clinically important developmental pathway. The Toxoplasma biology that underlies human chronic infection is developmental conversion of the acute tachyzoite stage into the latent bradyzoite stage. We investigated the roles of two alkaline-stress-induced ApiAP2 transcription factors, AP2IV-3 and AP2IX-9, in bradyzoite development. These factors were expressed in two overlapping waves during bradyzoite development, with AP2IX-9 increasing expression earlier than AP2IV-3, which peaked as AP2IX-9 expression was declining. Disruption of the AP2IX-9 gene enhanced, while deletion of AP2IV-3 gene decreased, tissue cyst formation, demonstrating that these factors have opposite functions in bradyzoite development. Conversely, conditional overexpression of FKBP-modified AP2IX-9 or AP2IV-3 with the small molecule Shield 1 had a reciprocal effect on tissue cyst formation, confirming the conclusions of the knockout experiments. The AP2IX-9 repressor and AP2IV-3 activator tissue cyst phenotypes were borne out in gene expression studies that determined that many of the same bradyzoite genes were regulated in an opposite manner by these transcription factors. A common gene target was the canonical bradyzoite marker BAG1, and mechanistic experiments determined that, like AP2IX-9, AP2IV-3 regulates a BAG1 promoter-luciferase reporter and specifically binds the BAG1 promoter in parasite chromatin. Altogether, these results suggest that the AP2IX-9 transcriptional repressor and the AP2IV-3 transcriptional activator likely compete to control bradyzoite gene expression, which may permit Toxoplasma to better adapt to different tissue environments and select a suitable host cell for long-term survival of the dormant tissue cyst. IMPORTANCE Toxoplasma infections are lifelong because of the development of the bradyzoite tissue cyst, which is effectively invisible to the immune system. Despite the important clinical consequences of this developmental pathway, the molecular basis of the switch mechanisms that control tissue cyst formation is still poorly understood. Significant changes in gene expression are associated with tissue cyst development, and ApiAP2 transcription factors are an important mechanism regulating this developmental transcriptome. However, the molecular composition of these ApiAP2 complexes and the operating principles of ApiAP2 mechanisms are not well defined. Here we establish that competing ApiAP2 transcriptional mechanisms operate to regulate this clinically important developmental pathway.

scholarly journals Toxoplasma gondiiAP2IX-4 regulates gene expression during bradyzoite development

2017 ◽  
Author(s):  
Sherri Huang ◽  
Michael J. Holmes ◽  
Joshua B. Radke ◽  
Dong-Pyo Hong ◽  
Ting-Kai Liu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Animal Health ◽  
Protozoan Parasite ◽  
Cyst Formation ◽  
Tissue Cyst ◽  
Alkaline Media ◽  
Alkaline Stress ◽  
Stress Induction ◽  
Reduced Frequency ◽  
Current Therapies

ABSTRACTToxoplasma gondiiis a protozoan parasite of great importance to human and animal health. In the host, this obligate intracellular parasite persists as a tissue cyst form that is invisible to the immune response and unaffected by current therapies. The tissue cysts facilitate transmission through predation and give rise to chronic cycles of toxoplasmosis in immune compromised patients. Transcriptional changes accompany conversion of the rapidly replicating tachyzoites into the encysted bradyzoites, yet the mechanisms underlying these alterations in gene expression are not well-defined. Here we show that AP2IX-4 is a nuclear protein exclusively expressed in tachyzoites and bradyzoites undergoing division. Knockout of AP2IX-4 had no discernible effect on tachyzoite replication, but resulted in a reduced frequency of tissue cyst formation following alkaline stress induction – a defect that is reversible by complementation. AP2IX-4 has a complex role in regulating bradyzoite gene expression, as many bradyzoite mRNAs dramatically increased beyond normal stress-induction in AP2IX-4 knockout parasites exposed to alkaline media. The loss of AP2IX-4 also resulted in a modest virulence defect and reduced cyst burden in chronically infected mice, which was also reversed by complementation. These findings illustrate that the transcriptional mechanisms responsible for tissue cyst development operate across the intermediate life cycle from the dividing tachyzoite to the dormant bradyzoite.

scholarly journals Transcriptional repression by ApiAP2 factors is central to chronic toxoplasmosis

2017 ◽  
Author(s):  
Joshua B. Radke ◽  
Danielle Worth ◽  
Dong-Pyo Hong ◽  
Sherri Huang ◽  
William J. Sullivan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcriptional Repression ◽  
Chronic Infection ◽  
Parasitophorous Vacuole ◽  
Acute Infection ◽  
Cyst Formation ◽  
Tissue Cyst ◽  
Genetic Lineages ◽  
Inflammatory Monocytes ◽  
Bradyzoite Differentiation

AbstractBradyzoite differentiation is marked by major changes in gene expression resulting in a parasite that expresses a new repertoire of surface antigens hidden inside a modified parasitophorous vacuole called the tissue cyst. The factors that control this important life cycle transition are not well understood. Here we describe an importantToxoplasmatranscriptional repressor mechanism controlling bradyzoite differentiation that operates exclusively in the tachyzoite stage. The ApiAP2 factor, AP2IV-4, is a nuclear factor dynamically expressed in late S phase through mitosis/cytokinesis of the tachyzoite cell cycle. Remarkably, deletion of the AP2IV-4 locus resulted in the increased expression of bradyzoite mRNAs in replicating tachyzoites, and in two different genetic lineages we confirmed the misexpression of tissue cyst wall components (e.g. BPK1, MCP4, CST1) and the bradyzoite surface antigen SRS9 in the tachyzoite stage. In the murine animal model, the loss of AP2IV-4 had profound biological consequences. Type II prugniaud strain parasites lacking AP2IV-4 were unable to form tissue cysts in brain tissue and the absence of this factor also recruited a potent immune response characterized by increases inflammatory monocytes, IFN-γ and higher numbers of both CD8+ and CD4+ T-cells. Altogether, these results indicate that suppression of bradyzoite antigens by AP2IV-4 during acute infection is required forToxoplasmato establish a chronic infection in the immune-competent host.Author SummaryTheToxoplasmabiology that underlies the establishment of a chronic infection is developmental conversion of the acute tachyzoite stage into the latent bradyzoite-tissue cyst stage. Despite the important clinical consequences of this developmental pathway, the molecular basis of the switch mechanisms that control formation of the tissue cyst is still poorly understood. A fundamental feature of tissue cyst formation is the expression of bradyzoite-specific genes. Here we show the transcription factor AP2IV-4 directly silences bradyzoite mRNA and protein expression in the acute tachyzoite stage demonstrating that developmental control of tissue cyst formation is as much about when not to express bradyzoite genes as it is about when to activate them. Loosing the suppression of bradyzoite gene expression in the acute tachyzoite stage caused by deleting AP2IV-4 blocked the establishment of chronic disease in healthy animals through the pre-arming of the immune system suggesting a possible strategy for preventing chronicToxoplasmainfections.

scholarly journals Toxoplasma gondii AP2IX-4 Regulates Gene Expression during Bradyzoite Development

mSphere ◽  
2017 ◽  
Vol 2 (2) ◽  
Author(s):  
Sherri Huang ◽  
Michael J. Holmes ◽  
Joshua B. Radke ◽  
Dong-Pyo Hong ◽  
Ting-Kai Liu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Toxoplasma Gondii ◽  
Cyst Formation ◽  
Tissue Cyst ◽  
Stress Induction ◽  
Content Type ◽  
A Cell ◽  
Transitional Forms ◽  
Mouse Model Of Infection ◽  
Cyst Development

ABSTRACT Toxoplasma gondii is a single-celled parasite that persists in its host as a transmissible tissue cyst. How the parasite converts from its replicative form to the bradyzoites housed in tissue cysts is not well understood, but the process clearly involves changes in gene expression. Here we report that parasites lacking a cell cycle-regulated transcription factor called AP2IX-4 display reduced frequencies of tissue cyst formation in culture and in a mouse model of infection. Parasites missing AP2IX-4 lose the ability to regulate bradyzoite genes during tissue cyst development. Expressed in developing bradyzoites still undergoing division, AP2IX-4 may serve as a useful marker in the study of transitional forms of the parasite. Toxoplasma gondii is a protozoan parasite of great importance to human and animal health. In the host, this obligate intracellular parasite persists as a tissue cyst that is imperceptible to the immune response and unaffected by current therapies. The tissue cysts facilitate transmission through predation and give rise to chronic cycles of toxoplasmosis in immunocompromised patients. Transcriptional changes accompany conversion of the rapidly replicating tachyzoites into the encysted bradyzoites, and yet the mechanisms underlying these alterations in gene expression are not well defined. Here we show that AP2IX-4 is a nuclear protein exclusively expressed in tachyzoites and bradyzoites undergoing division. Knockout of AP2IX-4 had no discernible effect on tachyzoite replication but resulted in a reduced frequency of tissue cyst formation following alkaline stress induction—a defect that is reversible by complementation. AP2IX-4 has a complex role in regulating bradyzoite gene expression, as the levels of many bradyzoite mRNAs dramatically increased beyond those seen under conditions of normal stress induction in AP2IX-4 knockout parasites exposed to alkaline media. The loss of AP2IX-4 also resulted in a modest virulence defect and reduced cyst burden in chronically infected mice, which was reversed by complementation. These findings illustrate that the transcriptional mechanisms responsible for tissue cyst development operate across the intermediate life cycle from the dividing tachyzoite to the dormant bradyzoite. IMPORTANCE Toxoplasma gondii is a single-celled parasite that persists in its host as a transmissible tissue cyst. How the parasite converts from its replicative form to the bradyzoites housed in tissue cysts is not well understood, but the process clearly involves changes in gene expression. Here we report that parasites lacking a cell cycle-regulated transcription factor called AP2IX-4 display reduced frequencies of tissue cyst formation in culture and in a mouse model of infection. Parasites missing AP2IX-4 lose the ability to regulate bradyzoite genes during tissue cyst development. Expressed in developing bradyzoites still undergoing division, AP2IX-4 may serve as a useful marker in the study of transitional forms of the parasite.

Mechanisms of MEF2C-Dependent Transcription in Lymphoid Differentiation

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4341-4341
Author(s):  
Nikki R. Kong ◽  
Li Chai ◽  
Astar Winoto ◽  
Robert Tjian
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Cell Differentiation ◽  
B Cell ◽  
Progenitor Cells ◽  
Luciferase Reporter ◽  
B Cell Differentiation ◽  
Reporter Assays ◽  
B Lineage ◽  
Transcription Program

Abstract Hematopoiesis is a multi-step developmental process that requires an intricate coordination of signal relays and transcriptional regulation to give rise to all blood lineages in the organism. Hematopoietic stem/progenitor cells (HSPCs) can be driven to differentiate along three main lineages: myeloid, erythroid, and lymphoid. One of the earliest lineage decisions for HSPCs is whether to adopt the lymphoid or myeloid fate. Despite the discovery of several transcription factors required for different lineages of hematopoietic differentiation, the understanding of how gene expression allows HSPCs to adopt the lymphoid fate still remains incomplete. A study using an inducible hematopoietic-specific (Mx1-Cre) KO mouse line found that Myocyte Enhancer Factor 2C (MEF2C) is required for multi-potent HSPCs to differentiate into the lymphoid lineage (Stehling-Sun et al, 2009). However, the mechanisms of how MEF2C is activated and in turn, drives lymphoid fate specification are not known. Through a candidates approach with co-expression and co-immunoprecipitation, we have identified Early B Cell Factor 1 (EBF1) to be a specific interacting partner of MEF2C, and not other B cell specific transcription factors such as E12, E47, or PAX5. Genome-wide survey of MEF2C and EBF1 binding sites via chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) in a proB cell line revealed that these two sequence-specific transcription factors co-occupy the promoters and intragenic regions of many B cell specific genes such as Il7ra, Myb, Foxo1, Ets1, Ebf1 itself, and Pou2af1. Regulatory regions of Il7ra and Foxo1 derived from the ChIP-seq data, as well as an artificial enhancer containing trimerized MEF2C and EBF1 binding sites, were examined in luciferase reporter assays and found to be sufficient to drive transcription from a minimal reporter in 293T cells. Further, this activation was co-dependent on MEF2C and EBF1 expression. The functional relevance of MEF2C binding was further supported by gene expression analyses of MEF2C-regulated B lineage genes in Mx1-Cre Mef2c KO mice compared to WT mice. Consistent with ChIP-seq and luciferase reporter assays, Myb, Ebf1, Il7ra, and Foxo1 all had significantly decreased expression levels in MEF2C-null HSPCs as well as B lineage progenitor cells, compared to sex-matched littermate control mice. Interestingly, myeloid gene expression in Mef2c-KO mice was increased compared to WT control. MEF2C ChIP-seq in a murine HSPC line revealed that it binds myeloid lineage gene targets that are not regulated by MEF2C in proB cells. These results suggest that MEF2C can repress myeloid gene expression in HSPCs. To elucidate the mechanism of this functional switch, we tested the requirement for MAPK pathways to phosphorylate and activate MEF2C at three previously identified residues in order to drive B cell differentiation. Inhibition of p38 MAPK (p38i), but not ERK1/2/5, decreased the potential of HSPCs to differentiate into B220+CD19+ B cells cultured with cytokines that drive this particular lineage fate. Instead, p38i-treated progenitor cells gave rise to more myeloid cells. 65% of this phenotype was rescued by over-expressing a phosphomimetic mutant of MEF2C that can bypass p38 inhibition. Furthermore, MEF2C is known to bind class II HDAC proteins to repress gene expression, providing a possible mechanism for its repression of myeloid transcription program. Supporting this mechanism, phosphomimetic and HDAC-binding double mutant of MEF2C can rescue p38 inhibition phenotype almost 100%. Taken together, this study elucidated the molecular mechanisms of a key lymphoid-specific lineage fate determinant, MEF2C. We discovered that p38 MAPK converts MEF2C from a transcriptional repressor to an activator by phosphorylation in B cell specification, which can be applied to understanding other cell differentiation processes regulated by this important stress-induced signaling pathway. Furthermore, we identified MEF2C’s binding and co-activating partner EBF1, several novel B cell specific targets that it activates in proB cells, and a novel myeloid transcription program that it represses in hematopoietic progenitors. Therefore, these results expanded our understanding of the intricate transcription network that regulates B cell differentiation. Disclosures No relevant conflicts of interest to declare.

scholarly journals Regulation of basal promoter activity of the human thiamine pyrophosphate transporter SLC44A4 in human intestinal epithelial cells

2015 ◽  
Vol 308 (9) ◽  
pp. C750-C757 ◽  
Author(s):  
Svetlana M. Nabokina ◽  
Mel Brendan Ramos ◽  
Judith E. Valle ◽  
Hamid M. Said
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Epithelial Cells ◽  
Promoter Activity ◽  
Minimal Promoter ◽  
Thiamine Pyrophosphate ◽  
Luciferase Reporter ◽  
Uptake System ◽  
Colonic Epithelial Cells ◽  
Basal Promoter Activity

Microbiota of the large intestine synthesize considerable amount of vitamin B1 in the form of thiamine pyrophosphate (TPP). There is a specific high-affinity regulated carrier-mediated uptake system for TPP in human colonocytes (product of the SLC44A4 gene). The mechanisms of regulation of SLC44A4 gene expression are currently unknown. In this study, we characterized the SLC44A4 minimal promoter region and identified transcription factors important for basal promoter activity in colonic epithelial cells. The 5′-regulatory region of the SLC44A4 gene (1,022 bp) was cloned and showed promoter activity upon transient transfection into human colonic epithelial NCM460 cells. With the use of a series of 5′- and 3′-deletion luciferase reporter constructs, the minimal genomic region that required basal transcription of the SLC44A4 gene expression was mapped between nucleotides −178 and +88 (using the distal transcriptional start site as +1). Mutational analysis performed on putative cis-regulatory elements established the involvement of ETS/ELF3 [E26 transformation-specific sequence (ETS) proteins], cAMP-responsive element (CRE), and SP1/GC-box sequence motifs in basal SLC44A4 promoter activity. By means of EMSA, binding of ELF3 and CRE-binding protein-1 (CREB-1) transcription factors to the SLC44A4 minimal promoter was shown. Contribution of CREB into SLC44A4 promoter activity was confirmed using NCM460 cells overexpressing CREB. We also found high expression of ELF3 and CREB-1 in colonic (NCM460) compared with noncolonic (ARPE19) cells, suggesting their possible contribution to colon-specific pattern of SLC44A4 expression. This study represents the first characterization of the SLC44A4 promoter and reports the importance of both ELF3 and CREB-1 transcription factors in the maintenance of basal promoter activity in colonic epithelial cells.

The Prepropalustrin-2CE2 and Preprobrevinin-2CE3　Gene from Rana　Chensinensis: Gene Expression, Genomic Organization, and Functional Analysis of the Promoter Activity

2021 ◽  
Vol 28 ◽  
Author(s):  
Ruifen Zhang ◽  
Jing Gao ◽  
Hui Xie ◽  
Yan Sun ◽  
Yuan Zhang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Antimicrobial Peptides ◽  
Antimicrobial Peptide ◽  
Luciferase Reporter ◽  
Expression Levels ◽  
Environmental Stimuli ◽  
Rana Chensinensis ◽  
Peptide Gene ◽  
Gene Expression Levels

Background: Palustrin-2CE2 and brevinin-2CE3 are antimicrobial peptides from Rana chensinensis. In R. chensinensis tadpoles, the expression of prepropalustrin-2CE2 and preprobrevinin-2CE3 increased with the developmental stage. In addition, the expression of the two genes was dramatically upregulated with stimulation by Escherichia coli, Staphylococcus aureus, and the chemical lipopolysaccharide (LPS). The genomic organization of the two antimicrobial peptide genes was confirmed. Both prepropalustrin-2CE2 and preprobrevinin-2CE3 contain three exons separated by two large introns. Additionally, several presumed transcription factor binding sites were identified in the promoter sequence. Functional analysis of the promoter was performed using a luciferase reporter system, and further confirmed by yeast one-hybrid experiment and EMSA assay. The results indicated that the transcription factors NF-κB and RelA are involved in regulating the expression of prepropalustrin-2CE2 and preprobrevinin-2CE3. As amphibian populations decline globally, this study provides new data demonstrating how frogs defend against pathogens from the environment by regulating AMP expression. For amphibians, antimicrobial peptides are innate immune molecules that resist adverse external environmental stimuli. However, the regulation mechanism of antimicrobial peptide gene expression in frogs is still unclear. Objective: The two antimicrobial peptides, palustrin-2CE2 and brevinin-2CE3, are produced under external stimulation in Rana chensinensis. Using this model, we analyzed the gene structure and regulatory elements of the two antimicrobial peptide genes and explored the regulatory effects of related transcription factors on the two genes. Method: Different stimuli such as E. coli, S. aureus, and chemical substance lipopolysaccharide (LPS) were applied to Rana chensinensis tadpoles at different developmental stages, and antimicrobial peptide expression levels were detected by RT-PCR. Bioinformatics analysis and 5'-RACE and genome walking technologies were employed to analyze the genome structure and promoter region of the antimicrobial peptide genes. With dual-luciferase reporter gene assays, yeast one-hybrid experiment and EMSA assays, we assessed the regulatory effect of the endogenous regulators of the cell on the antimicrobial peptide promoter. Results: The transcription levels of prepropalustrin-2CE2 and preprobrevinin-2CE3 were significantly upregulated after different stimulations. Genomic structure analysis showed that both genes contained three exons and two introns. Promoter analysis indicated that there are binding sites for regulatory factors of the NF-κB family in the promoter region, and experiments showed that endogenous NF-κB family regulatory factors in frog cells activate the promoters of the antimicrobial peptide genes. Yeast one-hybrid experiment and EMSA assay demonstrated that RelA and NF-κB1 might interact with specific motifs in the prepropalustrin-2CE2 promoter. Conclusion: In this paper, we found that the gene expression levels of the antimicrobial peptides, palustrin-2CE2 and brevinin-2CE3, in R. chensinensis will increase under environmental stimuli, and we verified that the changes in gene expression levels are affected by the transcription factors RelA and NF-κB1. The yeast one-hybrid experiment and EMSA assay confirmed that RelA and NF-κB1 could directly interact with the frog antimicrobial peptide gene promoter, providing new data for the regulatory mechanism of antimicrobial peptides in response to environmental stimuli.

FLT3ITD Target Enhancers Are Primed but Inaccessible in Fetal Hematopoietic Progenitors

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1228-1228
Author(s):  
Yanan Li ◽  
Riddhi M Patel ◽  
Emily Casey ◽  
Jeffrey A. Magee
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Target Gene ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Gene Activation ◽  
Chromatin Accessibility ◽  
Luciferase Reporter ◽  
Enhancer Activity ◽  
Target Gene Expression

The FLT3 Internal Tandem Duplication (FLT3ITD) is common somatic mutation in acute myeloid leukemia (AML). We have previously shown that FLT3ITD fails to induce changes in HSC self-renewal, myelopoiesis and leukemogenesis during fetal stages of life. FLT3ITD signal transduction pathways are hyperactivated in fetal progenitors, but FLT3ITD target genes are not. This suggests that postnatal-specific transcription factors may be required to help induce FLT3ITD target gene expression. Alternatively, repressive histone modifications may impose a barrier to FLT3ITD target gene activation in fetal HPCs that is relaxed during postnatal development. To resolve these possibilities, we used ATAC-seq, as well as H3K4me1, H3K27ac and H3K27me3 ChIP-seq, to identify cis-elements that putatively control FLT3ITD target gene expression in fetal and adult hematopoietic progenitor cells (HPCs). We identified many enhancer elements (ATAC-seq peaks with H3K4me1 and H3K27ac) that exhibited increased chromatin accessibility and activity in FLT3ITD adult HPCs relative to wild type adult HPCs. These elements were enriched near FLT3ITD target genes. HOMER analysis showed enrichment for STAT5, ETS, RUNX1 and IRF binding motifs within the FLT3ITD target enhancers, but motifs for temporally dynamic transcription factors were not identified. We cloned a subset of the enhancers and confirmed that they could synergize with their promoter to activate a luciferase reporter. For representative enhancers, STAT5 binding sites were required to activate the enhancer - as anticipated - and RUNX1 repressed enhancer activity. We tested whether accessibility or priming changed between fetal and adult stages of HPC development. FLT3ITD-dependent changes in chromatin accessibility were not observed in fetal HPCs, though the enhancers were primed early in development as evidenced by the presence of H3K4me1. Repressive H3K27me3 were not present at FLT3ITD target enhancers in either or adult HPCs. The data show that FLT3ITD target enhancers are demarcated early in hematopoietic development, long before they become responsive to FLT3ITD signaling. Repressive marks do not appear to create an epigenetic barrier to enhancer activation in the fetal stage. Instead, age-specific transcription factors are likely required to pioneer enhancer elements so that they can respond to STAT5 and other FLT3ITD effectors. Disclosures No relevant conflicts of interest to declare.

scholarly journals Expression of the human oestrogen receptor-alpha gene is regulated by promoter F in MG-63 osteoblastic cells

2003 ◽  
Vol 372 (3) ◽  
pp. 831-839 ◽  
Author(s):  
Elisabetta LAMBERTINI ◽  
Letizia PENOLAZZI ◽  
Silvia GIORDANO ◽  
Laura DEL SENNO ◽  
Roberta PIVA
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Transcription Start Site ◽  
Thyroid Hormone Receptor ◽  
Regulatory Elements ◽  
Luciferase Reporter ◽  
Start Site ◽  
Mcf7 Cells ◽  
Transcription Start ◽  
Specific Element

(O)estrogen receptor-α (ERα), a hormone-dependent transcription factor belonging to the steroid/thyroid-hormone-receptor superfamily, plays an essential role in the development and maintenance of the skeleton. Here we report the analysis of an unexplored sequence inside the bone-specific distal promoter F (PF) with respect to the regulation of ERα gene expression in bone. This sequence, 785 bp in size, is localized upstream of the assigned transcription start site of exon F, at −117140 bp from the originally described transcription start site +1. It contains a TA reach box, a conventional CAAT box and potential regulatory elements for many transcription factors, including Cbfa1 [OSE2 (osteoblast-specific element) core binding factor], GATA-1 [(A/T)GATA(A/G) binding protein], Sox5 [sex-determining region Y (SRY)-type HMG bOX protein, belonging to a subfamily of DNA-binding proteins with an HMG domain], Sry, AP1 (activator protein 1) and CP2 (activator of γ-globin). It is able to strongly activate the luciferase reporter gene in MG-63 osteoblastic-like cells, but not in MCF7 breast-cancer cells. This is in agreement with different transcripts that we found in the two cell types. The footprinting and electrophoretic mobility-shift assays (EMSAs) showed that, inside the region analysed, there were some sequences that specifically reacted to nuclear proteins isolated from MG-63 cells. In particular, we identified two regions, named PFa and PFb, that do not present binding sites for known transcription factors and that are involved in a strong DNA–protein interaction in MG-63, but not in MCF7, cells. The analysis of three transcription factors (GATA-1, Sry and Sox) that might bind the identified footprinted areas suggested a possible indirect role of these proteins in the regulation of ERα gene expression in bone. These data provide evidence for different promoter usage of the ERα gene through the recruitment of tissue-specific transcription activators and co-regulators.

Use of Transgenic Luciferase Reporter Mice To Determine Activation of Transcription Factors and Gene Expression by Fibrogenic Particles

CHEST Journal ◽  
2001 ◽  
Vol 120 (1) ◽  
pp. S24-S25 ◽  
Author(s):  
Andrea K. Hubbard ◽  
Cynthia R. Timblin ◽  
Mercedes Rincon ◽  
Brooke T. Mossman
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Luciferase Reporter ◽  
Reporter Mice ◽  
Activation Of Transcription
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