Abstract P415: Etv2 Interacts With Vezf1 And Co-regulate Endothelial Gene Expression

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Satyabrata Das ◽  
Vinayak Gupta ◽  
Wuming Gong ◽  
Javier Sierra-pagan ◽  
Erik Skie ◽  
...  
Keyword(s):  
Candidate Genes ◽  
Cell Fate ◽  
Zinc Finger ◽  
Target Genes ◽  
Embryoid Bodies ◽  
Mobility Shift ◽  
Histone Acetyl Transferase ◽  
Critical Function ◽  
Binding Partner ◽  
Endothelial Development

Background: Ets transcription factors function as important developmental regulators and are known to be modulators of cell fate. Previously, in conjunction with co-factors, we have described Ets variant 2 (Etv2) as an essential regulator of the hematopoietic and endothelial lineages. But the mechanism and the Etv2 interacting partners involved in achieving this critical function remains poorly understood. Results: Using a yeast two-hybrid assay, we identified Vascular Endothelial Zinc Finger 1 (Vezf1) as an interacting factor with Etv2. Vezf1 is a conserved C2H2 zinc finger transcription factor known to regulate the formation, proliferation, and migration of endothelial cells. We verified Vezf1 as a binding partner of Etv2 through co-immunoprecipitation and GST-pull down studies. Bioinformatics analysis of ChIP-seq and Etv2-expressing single cell RNA sequencing was conducted to identify candidate genes containing both Etv2 and Vezf1 binding motifs in their regulatory regions. Histone deacetylase 7 (Hdac7) and angiomotin like protein 2 (Amotl2) were identified as candidate genes and have previously been shown to function as regulators of endothelial development. RT-qPCR analysis showed upregulation of Hdac7 and Amotl2 in response to doxycycline inducible Etv2 and Vezf1; whereas significant reduction of expression of these two genes was observed in the Etv2 and Vezf1 knockout cells. Chromatin immunoprecipitation (ChIP) and electrophoresis mobility shift assays (EMSA) confirmed Etv2-Vezf1 adjacent binding sites in the promoters of Hdac7 and Amotl2. Histone Acetyl transferase (HAT) assays was performed to investigate Etv2-Vezf1 on global histone acetylation conditions in doxycycline inducible embryoid bodies. Vezf1 overexpression results in a significant reduction of acetylated histones. Additionally, we have undertaken ATAC-Seq (Assay for Transposase-Accessible Chromatin using sequencing) and Vezf1 ChIP-Seq studies to profile the epigenetic modifications and genome-wide occupancy of Vezf1 in the presence or absence of Etv2 in endothelial progenitor cells. Conclusion: In summary, this study identifies Vezf1 as a novel binding partner of Etv2 and we further demonstrate their combined role in the regulation of downstream target genes, Amotl2 and Hdac7, during endothelial development.

scholarly journals The transcription factors Scl and Lmo2 act together during development of the hemangioblast in zebrafish

Blood ◽  
2006 ◽  
Vol 109 (6) ◽  
pp. 2389-2398 ◽  
Author(s):  
Lucy J. Patterson ◽  
Martin Gering ◽  
Craig E. Eckfeldt ◽  
Anthony R. Green ◽  
Catherine M. Verfaillie ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Target Genes ◽  
Embryoid Bodies ◽  
Specific Marker ◽  
Zebrafish Embryos ◽  
Binding Partners ◽  
Binding Forms ◽  
Endothelial Development ◽  
Upstream Regulators

Abstract The transcription factors Scl and Lmo2 are crucial for development of all blood. An important early requirement for Scl in endothelial development has also been revealed recently in zebrafish embryos, supporting previous findings in scl−/− embryoid bodies. Scl depletion culminates most notably in failure of dorsal aorta formation, potentially revealing a role in the formation of hemogenic endothelium. We now present evidence that the requirements for Lmo2 in zebrafish embryos are essentially the same as for Scl. The expression of important hematopoietic regulators is lost, reduced, or delayed, panendothelial gene expression is down-regulated, and aorta-specific marker expression is lost. The close similarity of the phenotypes for Scl and Lmo2 suggest that they perform these early functions in hemangioblast development within a multiprotein complex, as shown for erythropoiesis. Consistent with this, we find that scl morphants cannot be rescued by a non-Lmo2–binding form of Scl but can be rescued by non-DNA–binding forms, suggesting tethering to target genes through DNA-binding partners linked via Lmo2. Interestingly, unlike other hematopoietic regulators, the Scl/Lmo2 complex does not appear to autoregulate, as neither gene's expression is affected by depletion of the other. Thus, expression of these critical regulators is dependent on continued expression of upstream regulators, which may include cell-extrinsic signals.

scholarly journals The H3K9 methyltransferase SETDB1 maintains female identity in Drosophila germ cells by repressing expression of key spermatogenesis genes

2018 ◽  
Author(s):  
Anne E. Smolko ◽  
Laura Shapiro-Kulnane ◽  
Helen K. Salz
Keyword(s):  
Germ Cell ◽  
Cell Fate ◽  
Germ Cells ◽  
Target Genes ◽  
Female Identity ◽  
Binding Partner ◽  
Regional Deposition ◽  
Female Specific ◽  
Inappropriate Expression ◽  
Novel Model

AbstractThe preservation of germ cell sexual identity is essential for gametogenesis. Here we show that H3K9me3-mediated gene silencing is integral to female fate maintenance in Drosophila germ cells. Germ cell-specific loss of the H3K9me3 pathway members, the trimethyltransferase SETDB1, its binding partner WDE, and the H3K9 binding protein HP1a, cause the inappropriate expression of testis genes. SETDB1 is required for H3K9me3 accumulation on a select subset of the silenced testis genes. Interestingly, these SETDB1-dependent H3K9me3 domains are highly localized and do not spread into neighboring loci. Regional deposition is especially striking at the phf7 locus, a key regulator of male germ cell sexual fate. phf7 is primarily regulated by alternative promoter usage and transcription start site (TSS) selection. We find H3K9me3 accumulation is restricted to the silenced testis-specific TSS region in ovaries. Furthermore, its recruitment to phf7 and repression of the testis-specific transcript is dependent on the female sex determination gene Sxl. These findings demonstrate that female identity is secured by a pathway in which Sxl is the upstream female-specific regulator, SETDB1 is the required chromatin writer and phf7 is one of the critical SETDB1 target genes. This function of SETDB1 is unrelated to its canonical role in piRNA biogenesis and silencing of transposable elements. Collectively our findings support a novel model in which female fate is preserved by deposition of H3K9me3 repressive marks on key spermatogenesis genes and suggest that this strategy for securing cell fate may be widespread.

Pbx-1 Is a Direct Target of Evi-1 in Hematopoietic Stem/Progenitors and Leukemic Cells.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1192-1192
Author(s):  
Munetake Shimabe ◽  
Susumu Goyama ◽  
Motoshi Ichikawa ◽  
Yoichi Imai ◽  
Tsuyoshi Takahashi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Candidate Genes ◽  
Zinc Finger ◽  
Target Genes ◽  
Leukemic Cells ◽  
Hematopoietic Stem ◽  
Direct Target

Abstract The ecotropic viral integration site-1 (Evi-1) gene was first identified as a common locus of retroviral integration in murine leukemia. In humans, Evi-1 is located on chromosome 3q26, and rearrangements on chrmosome 3q26 often activate Evi-1 expression in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Patients with these karyotypes are characterized by the elevated platelet count and lack of response to antileukemic therapy. Elevated Evi-1 expression occurs with high frequency in AML patients without 3q26 abnormalities, and is also associated with unfavorable outcomes. Thus, Evi-1 is one of the key factors that predict poor survival in leukemia patients. Evi-1 is a member of the SET/PR domain family of transcription factors and it contains a total of 10 zinc finger motifs organized in two discrete domains, comprising 7 and 3 repeats respectively, which have distinct DNA binding specificities. Recently, we generated Evi-1-mutant mice and showed that Evi-1 plays an essential role in proliferation of both hematopoietic stem cells (HSCs) and transformed leukemic cells. Furthermore, we identified candidate target genes of Evi-1 using gene expression profiling analysis in HSCs combined with the gene expression data of AML samples. These genes include Gata1, Gata2, Angpt1, Mpl, Jag2, Pbx-1, Setbp1 and Itga2b. In this study, we first analyzed relative gene expression of these candidate genes in control- or Evi-1-transduced hematopoietic stem/progenitors (c-Kit+ cells). Among these candidate genes, Evi-1 up-regulates transcription of Pbx-1, Mpl, Setbp1 and Itga2b. Next, we cloned 5 ′ up-stream genomic regions of these four genes into the pGL-4 luciferase reporter vector, and found that Evi-1 increased luciferase activity of Pbx-1 reporter construct in COS7 cells. Deletion of reporter constructs revealed that Evi-1 binds to -0.5kb upstream of the transcription start site of Pbx-1. We then examined the transcription activity of a series of Evi-1 mutants and found that both the first and second zinc finger domains are required for the Pbx-1 up-regulation. Furthermore, chromatin immunoprecipitation (ChIP) analysis revealed that Evi-1 directly binds to the promoter region of Pbx-1. We next evaluated a role for Pbx-1 in Evi-1-induced myeloid transformation. Bone marrow progenitors transduced with Evi-1 showed sustained colony formation in the serial replating assay. After establishment of sustained clonogenic activity following more than three rounds of replating in methylcellulose medium, the cells were transduced with control or Pbx-1-shRNA. Interestingly, reduction of Pbx-1 levels through RNAi-mediated knockdown significantly inhibited Evi-1-induced transformation. In contrast, knockdown of Pbx-1 did not impair bone marrow transformation by transcription factor E2A-hepatic leukemia factor (E2A-HLF), suggesting that Pbx-1 is specifically, as opposed to generally, required for maintenance of transformation mediated by Evi-1. Taken together, these results indicate that Pbx-1 is one of the direct target genes of Evi- 1 in hematopoietic cells, and aberrant Pbx-1 expression is responsible, at least in part, for the oncogenic activity of Evi-1. Because Pbx-1 is known as a critical regulator of hematopoietic stem cells and leukemia development, the Evi-1-Pbx-1 pathway may be a key modulator of stem cell activity in normal and malignant hematopoiesis.

scholarly journals Chromatin Manipulation and Editing: Challenges, New Technologies and Their Use in Plants

2021 ◽  
Vol 22 (2) ◽  
pp. 512
Author(s):  
Kateryna Fal ◽  
Denisa Tomkova ◽  
Gilles Vachon ◽  
Marie-Edith Chabouté ◽  
Alexandre Berr ◽  
...  
Keyword(s):  
Cell Fate ◽  
Dna Sequence ◽  
Zinc Finger ◽  
Chromatin Structure ◽  
New Technologies ◽  
Dna Interaction ◽  
The Other ◽  
Proof Of Concept ◽  
Epigenetic Marks ◽  
Molecular Events

An ongoing challenge in functional epigenomics is to develop tools for precise manipulation of epigenetic marks. These tools would allow moving from correlation-based to causal-based findings, a necessary step to reach conclusions on mechanistic principles. In this review, we describe and discuss the advantages and limits of tools and technologies developed to impact epigenetic marks, and which could be employed to study their direct effect on nuclear and chromatin structure, on transcription, and their further genuine role in plant cell fate and development. On one hand, epigenome-wide approaches include drug inhibitors for chromatin modifiers or readers, nanobodies against histone marks or lines expressing modified histones or mutant chromatin effectors. On the other hand, locus-specific approaches consist in targeting precise regions on the chromatin, with engineered proteins able to modify epigenetic marks. Early systems use effectors in fusion with protein domains that recognize a specific DNA sequence (Zinc Finger or TALEs), while the more recent dCas9 approach operates through RNA-DNA interaction, thereby providing more flexibility and modularity for tool designs. Current developments of “second generation”, chimeric dCas9 systems, aiming at better targeting efficiency and modifier capacity have recently been tested in plants and provided promising results. Finally, recent proof-of-concept studies forecast even finer tools, such as inducible/switchable systems, that will allow temporal analyses of the molecular events that follow a change in a specific chromatin mark.

scholarly journals Linking metabolic dysfunction with cardiovascular diseases: Brn-3b/POU4F2 transcription factor in cardiometabolic tissues in health and disease

2021 ◽  
Vol 12 (3) ◽  
Author(s):  
Vishwanie S. Budhram-Mahadeo ◽  
Matthew R. Solomons ◽  
Eeshan A. O. Mahadeo-Heads
Keyword(s):  
Transcription Factor ◽  
Cardiovascular Diseases ◽  
Cell Fate ◽  
Target Genes ◽  
Cardiovascular Responses ◽  
Normal Function ◽  
Valuable Insight ◽  
Metabolic Dysfunction ◽  
Pathological Changes ◽  
Cardiac Responses

AbstractMetabolic and cardiovascular diseases are highly prevalent and chronic conditions that are closely linked by complex molecular and pathological changes. Such adverse effects often arise from changes in the expression of genes that control essential cellular functions, but the factors that drive such effects are not fully understood. Since tissue-specific transcription factors control the expression of multiple genes, which affect cell fate under different conditions, then identifying such regulators can provide valuable insight into the molecular basis of such diseases. This review explores emerging evidence that supports novel and important roles for the POU4F2/Brn-3b transcription factor (TF) in controlling cellular genes that regulate cardiometabolic function. Brn-3b is expressed in insulin-responsive metabolic tissues (e.g. skeletal muscle and adipose tissue) and is important for normal function because constitutive Brn-3b-knockout (KO) mice develop profound metabolic dysfunction (hyperglycaemia; insulin resistance). Brn-3b is highly expressed in the developing hearts, with lower levels in adult hearts. However, Brn-3b is re-expressed in adult cardiomyocytes following haemodynamic stress or injury and is necessary for adaptive cardiac responses, particularly in male hearts, because male Brn-3b KO mice develop adverse remodelling and reduced cardiac function. As a TF, Brn-3b regulates the expression of multiple target genes, including GLUT4, GSK3β, sonic hedgehog (SHH), cyclin D1 and CDK4, which have known functions in controlling metabolic processes but also participate in cardiac responses to stress or injury. Therefore, loss of Brn-3b and the resultant alterations in the expression of such genes could potentially provide the link between metabolic dysfunctions with adverse cardiovascular responses, which is seen in Brn-3b KO mutants. Since the loss of Brn-3b is associated with obesity, type II diabetes (T2DM) and altered cardiac responses to stress, this regulator may provide a new and important link for understanding how pathological changes arise in such endemic diseases.

scholarly journals Decoding the molecular mechanism of parthenocarpy in Musa spp. through protein–protein interaction network

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Suthanthiram Backiyarani ◽  
Rajendran Sasikala ◽  
Simeon Sharmiladevi ◽  
Subbaraya Uma
Keyword(s):  
Candidate Genes ◽  
Protein Interaction ◽  
Target Genes ◽  
Interaction Network ◽  
Enrichment Analysis ◽  
Auxin Signaling ◽  
Pathway Enrichment Analysis ◽  
Ppi Network ◽  
Protein Protein Interaction ◽  
The Difference

AbstractBanana, one of the most important staple fruit among global consumers is highly sterile owing to natural parthenocarpy. Identification of genetic factors responsible for parthenocarpy would facilitate the conventional breeders to improve the seeded accessions. We have constructed Protein–protein interaction (PPI) network through mining differentially expressed genes and the genes used for transgenic studies with respect to parthenocarpy. Based on the topological and pathway enrichment analysis of proteins in PPI network, 12 candidate genes were shortlisted. By further validating these candidate genes in seeded and seedless accession of Musa spp. we put forward MaAGL8, MaMADS16, MaGH3.8, MaMADS29, MaRGA1, MaEXPA1, MaGID1C, MaHK2 and MaBAM1 as possible target genes in the study of natural parthenocarpy. In contrary, expression profile of MaACLB-2 and MaZEP is anticipated to highlight the difference in artificially induced and natural parthenocarpy. By exploring the PPI of validated genes from the network, we postulated a putative pathway that bring insights into the significance of cytokinin mediated CLAVATA(CLV)–WUSHEL(WUS) signaling pathway in addition to gibberellin mediated auxin signaling in parthenocarpy. Our analysis is the first attempt to identify candidate genes and to hypothesize a putative mechanism that bridges the gaps in understanding natural parthenocarpy through PPI network.

scholarly journals From cytopenia to leukemia: the role of Gfi1 and Gfi1b in blood formation

Blood ◽  
2015 ◽  
Vol 126 (24) ◽  
pp. 2561-2569 ◽  
Author(s):  
Tarik Möröy ◽  
Lothar Vassen ◽  
Brian Wilkes ◽  
Cyrus Khandanpour
Keyword(s):  
Zinc Finger ◽  
Target Genes ◽  
Integration Site ◽  
Wide Spectrum ◽  
Human Leukemia ◽  
Specific Expression ◽  
Hematopoietic Stem ◽  
Proviral Integration Site ◽  
Cell Type Specific Expression

AbstractThe DNA-binding zinc finger transcription factors Gfi1 and Gfi1b were discovered more than 20 years ago and are recognized today as major regulators of both early hematopoiesis and hematopoietic stem cells. Both proteins function as transcriptional repressors by recruiting histone-modifying enzymes to promoters and enhancers of target genes. The establishment of Gfi1 and Gfi1b reporter mice made it possible to visualize their cell type–specific expression and to understand their function in hematopoietic lineages. We now know that Gfi1 is primarily important in myeloid and lymphoid differentiation, whereas Gfi1b is crucial for the generation of red blood cells and platelets. Several rare hematologic diseases are associated with acquired or inheritable mutations in the GFI1 and GFI1B genes. Certain patients with severe congenital neutropenia carry mutations in the GFI1 gene that lead to the disruption of the C-terminal zinc finger domains. Other mutations have been found in the GFI1B gene in families with inherited bleeding disorders. In addition, the Gfi1 locus is frequently found to be a proviral integration site in retrovirus-induced lymphomagenesis, and new, emerging data suggest a role of Gfi1 in human leukemia and lymphoma, underlining the role of both factors not only in normal hematopoiesis, but also in a wide spectrum of human blood diseases.

Notch restricts lymphatic vessel sprouting induced by vascular endothelial growth factor

Blood ◽  
2011 ◽  
Vol 118 (4) ◽  
pp. 1154-1162 ◽  
Author(s):  
Wei Zheng ◽  
Tuomas Tammela ◽  
Masahiro Yamamoto ◽  
Andrey Anisimov ◽  
Tanja Holopainen ◽  
...  
Keyword(s):  
Notch Signaling ◽  
Cell Fate ◽  
Target Genes ◽  
Notch Pathway ◽  
Lymphatic Vessels ◽  
Soluble Form ◽  
Fibrin Gel ◽  
3 Dimensional ◽  
Endothelial Growth Factor

Abstract Notch signaling plays a central role in cell-fate determination, and its role in lateral inhibition in angiogenic sprouting is well established. However, the role of Notch signaling in lymphangiogenesis, the growth of lymphatic vessels, is poorly understood. Here we demonstrate Notch pathway activity in lymphatic endothelial cells (LECs), as well as induction of delta-like ligand 4 (Dll4) and Notch target genes on stimulation with VEGF or VEGF-C. Suppression of Notch signaling by a soluble form of Dll4 (Dll4-Fc) synergized with VEGF in inducing LEC sprouting in 3-dimensional (3D) fibrin gel assays. Expression of Dll4-Fc in adult mouse ears promoted lymphangiogenesis, which was augmented by coexpressing VEGF. Lymphangiogenesis triggered by Notch inhibition was suppressed by a monoclonal VEGFR-2 Ab as well as soluble VEGF and VEGF-C/VEGF-D ligand traps. LECs transduced with Dll4 preferentially adopted the tip cell position over nontransduced cells in 3D sprouting assays, suggesting an analogous role for Dll4/Notch in lymphatic and blood vessel sprouting. These results indicate that the Notch pathway controls lymphatic endothelial quiescence, and explain why LECs are poorly responsive to VEGF compared with VEGF-C. Understanding the role of the Notch pathway in lymphangiogenesis provides further insight for the therapeutic manipulation of the lymphatic vessels.

Myostatin regulates fiber-type composition of skeletal muscle by regulating MEF2 and MyoD gene expression

2009 ◽  
Vol 296 (3) ◽  
pp. C525-C534 ◽  
Author(s):  
Alex Hennebry ◽  
Carole Berry ◽  
Victoria Siriett ◽  
Paul O'Callaghan ◽  
Linda Chau ◽  
...  
Keyword(s):  
Growth Factor ◽  
Target Genes ◽  
Fiber Type ◽  
Fiber Formation ◽  
Type I ◽  
Mobility Shift ◽  
Enhancer Activity ◽  
Fiber Type Composition ◽  
Nuclear Extracts ◽  
Type Composition

Myostatin (Mstn) is a secreted growth factor belonging to the tranforming growth factor (TGF)-β superfamily. Inactivation of murine Mstn by gene targeting, or natural mutation of bovine or human Mstn, induces the double muscling (DM) phenotype. In DM cattle, Mstn deficiency increases fast glycolytic (type IIB) fiber formation in the biceps femoris (BF) muscle. Using Mstn null (−/−) mice, we suggest a possible mechanism behind Mstn-mediated fiber-type diversity. Histological analysis revealed increased type IIB fibers with a concomitant decrease in type IIA and type I fibers in the Mstn−/−tibialis anterior and BF muscle. Functional electrical stimulation of Mstn−/−BF revealed increased fatigue susceptibility, supporting increased type IIB fiber content. Given the role of myocyte enhancer factor 2 (MEF2) in oxidative type I fiber formation, MEF2 levels in Mstn−/−tissue were quantified. Results revealed reduced MEF2C protein in Mstn−/−muscle and myoblast nuclear extracts. Reduced MEF2-DNA complex was also observed in electrophoretic mobility-shift assay using Mstn−/−nuclear extracts. Furthermore, reduced expression of MEF2 downstream target genes MLC1F and calcineurin were found in Mstn−/−muscle. Conversely, Mstn addition was sufficient to directly upregulate MLC promoter-enhancer activity in cultured myoblasts. Since high MyoD levels are seen in fast fibers, we analyzed MyoD levels in the muscle. In contrast to MEF2C, MyoD levels were increased in Mstn−/−muscle. Together, these results suggest that while Mstn positively regulates MEF2C levels, it negatively regulates MyoD expression in muscle. We propose that Mstn could regulate fiber-type composition by regulating the expression of MEF2C and MyoD during myogenesis.

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