Abstract B222: Dicer substrate siRNAs to MYC, B-catenin, and other target genes effectively induce in vivo target gene knockdown and tumor inhibition.

Abstract Aims High-density lipoproteins (HDLs) are circulating micelles that transport proteins, lipids, and miRNAs. HDL-transported miRNAs (HDL-miRNAs) have lately received attention but their effects on vascular cells are not fully understood. Additionally, whether cardiovascular risk factors affect HDL-miRNAs levels and miRNA transfer to recipient cells remains equally poorly known. Here, we have investigated the changes induced by hypercholesterolaemia on HDL-miRNA levels and its effect on recipient endothelial cells (ECs). Methods and results Pigs were kept on a high-fat diet (HC; n = 10) or a normocholesterolaemic chow (NC; n = 10) for 10 days reaching cholesterol levels of 321.0 (229.7–378.5) mg/dL and 74.0 (62.5–80.2) mg/dL, respectively. HDL particles were isolated, purified, and quantified. HDL-miRNA profiling (n = 149 miRNAs) of HC- and NC-HDLs was performed by multipanel qPCR. Cell cultures of porcine aortic ECs were used to determine whether HDL-miRNAs were delivered to ECs. Potential target genes modulated by miRNAs were identified by bioinformatics and candidate miRNAs were validated by molecular analysis. In vivo effects in the coronary arteries of normocholesterolaemic swine administered HC- or NC-HDLs were analysed. Among the HDL-miRNAs, four were found in different amounts in HC- and NC-HDL (P < 0.05). miR-126-5p and -3p and miR-30b-5p (2.7×, 1.7×, and 1.3×, respectively) were found in higher levels and miR-103a-3p and miR-let-7g-5p (−1.6×, −1.4×, respectively) in lower levels in HC-HDL. miR-126-5p and -3p were transferred from HC-HDL to EC (2.5×; P < 0.05), but not from NC-HDL, by a SRB1-mediated mechanism. Bioinformatics revealed that HIF1α was the miR-126 target gene with the highest predictive value, which was accordingly found to be markedly reduced in HC-HDL-treated ECs and in miR126 mimic transfected ECs. In vivo validation confirmed that HIF1α was diminished in the coronary endothelial layer of NC pigs administered HC-HDL vs. those administered NC-HDL (P < 0.05). Conclusion Hypercholesterolaemia induces changes in the miRNA content of HDL enhancing miR126 and its delivery to ECs with the consequent down-regulation of its target gene HIF1α.

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Differential Contribution of N- and C-Terminal Regions of HIF1α and HIF2α to Their Target Gene Selectivity

International Journal of Molecular Sciences ◽

10.3390/ijms21249401 ◽

2020 ◽

Vol 21 (24) ◽

pp. 9401

Author(s):

Antonio Bouthelier ◽

Florinda Meléndez-Rodríguez ◽

Andrés A. Urrutia ◽

Julián Aragonés

Keyword(s):

Transcription Factors ◽

Dna Binding ◽

Cellular Response ◽

Target Gene ◽

Target Genes ◽

Transactivation Domain ◽

Transactivation Domains ◽

Relative Contribution

Cellular response to hypoxia is controlled by the hypoxia-inducible transcription factors HIF1α and HIF2α. Some genes are preferentially induced by HIF1α or HIF2α, as has been explored in some cell models and for particular sets of genes. Here we have extended this analysis to other HIF-dependent genes using in vitro WT8 renal carcinoma cells and in vivo conditional Vhl-deficient mice models. Moreover, we generated chimeric HIF1/2 transcription factors to study the contribution of the HIF1α and HIF2α DNA binding/heterodimerization and transactivation domains to HIF target specificity. We show that the induction of HIF1α-dependent genes in WT8 cells, such as CAIX (CAR9) and BNIP3, requires both halves of HIF, whereas the HIF2α transactivation domain is more relevant for the induction of HIF2 target genes like the amino acid carrier SLC7A5. The HIF selectivity for some genes in WT8 cells is conserved in Vhl-deficient lung and liver tissue, whereas other genes like Glut1 (Slc2a1) behave distinctly in these tissues. Therefore the relative contribution of the DNA binding/heterodimerization and transactivation domains for HIF target selectivity can be different when comparing HIF1α or HIF2α isoforms, and that HIF target gene specificity is conserved in human and mouse cells for some of the genes analyzed.

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A Multiplicity of Coactivators Is Required by Gcn4p at Individual Promoters In Vivo

Molecular and Cellular Biology ◽

10.1128/mcb.23.8.2800-2820.2003 ◽

2003 ◽

Vol 23 (8) ◽

pp. 2800-2820 ◽

Cited By ~ 108

Author(s):

Mark J. Swanson ◽

Hongfang Qiu ◽

Laarni Sumibcay ◽

Anna Krueger ◽

Soon-ja Kim ◽

...

Keyword(s):

Amino Acid ◽

Chromatin Immunoprecipitation ◽

Target Gene ◽

Target Genes ◽

Yeast Cells ◽

Histone Acetyltransferases ◽

Vitro Binding ◽

Paf1 Complex

ABSTRACT Transcriptional activators interact with multisubunit coactivators that modify chromatin structure or recruit the general transcriptional machinery to their target genes. Budding yeast cells respond to amino acid starvation by inducing an activator of amino acid biosynthetic genes, Gcn4p. We conducted a comprehensive analysis of viable mutants affecting known coactivator subunits from the Saccharomyces Genome Deletion Project for defects in activation by Gcn4p in vivo. The results confirm previous findings that Gcn4p requires SAGA, SWI/SNF, and SRB mediator (SRB/MED) and identify key nonessential subunits of these complexes required for activation. Among the numerous histone acetyltransferases examined, only that present in SAGA, Gcn5p, was required by Gcn4p. We also uncovered a dependence on CCR4-NOT, RSC, and the Paf1 complex. In vitro binding experiments suggest that the Gcn4p activation domain interacts specifically with CCR4-NOT and RSC in addition to SAGA, SWI/SNF, and SRB/MED. Chromatin immunoprecipitation experiments show that Mbf1p, SAGA, SWI/SNF, SRB/MED, RSC, CCR4-NOT, and the Paf1 complex all are recruited by Gcn4p to one of its target genes (ARG1) in vivo. We observed considerable differences in coactivator requirements among several Gcn4p-dependent promoters; thus, only a subset of the array of coactivators that can be recruited by Gcn4p is required at a given target gene in vivo.

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Identification of atrial fibrillation associated genes and functional non-coding variants

Nature Communications ◽

10.1038/s41467-019-12721-5 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 16

Author(s):

Antoinette F. van Ouwerkerk ◽

Fernanda M. Bosada ◽

Karel van Duijvenboden ◽

Matthew C. Hill ◽

Lindsey E. Montefiori ◽

...

Keyword(s):

Atrial Fibrillation ◽

Genetic Variants ◽

Target Gene ◽

Target Genes ◽

Association Studies ◽

Regulatory Region ◽

Regulatory Elements ◽

Genome Wide Association Studies ◽

Cx43 Expression

Abstract Disease-associated genetic variants that lie in non-coding regions found by genome-wide association studies are thought to alter the functionality of transcription regulatory elements and target gene expression. To uncover causal genetic variants, variant regulatory elements and their target genes, here we cross-reference human transcriptomic, epigenomic and chromatin conformation datasets. Of 104 genetic variant regions associated with atrial fibrillation candidate target genes are prioritized. We optimize EMERGE enhancer prediction and use accessible chromatin profiles of human atrial cardiomyocytes to more accurately predict cardiac regulatory elements and identify hundreds of sub-threshold variants that co-localize with regulatory elements. Removal of mouse homologues of atrial fibrillation-associated regions in vivo uncovers a distal regulatory region involved in Gja1 (Cx43) expression. Our analyses provide a shortlist of genes likely affected by atrial fibrillation-associated variants and provide variant regulatory elements in each region that link genetic variation and target gene regulation, helping to focus future investigations.

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Peroxisome Proliferator-Activated Receptor γ Target Gene Encoding a Novel Angiopoietin-Related Protein Associated with Adipose Differentiation

Molecular and Cellular Biology ◽

10.1128/mcb.20.14.5343-5349.2000 ◽

2000 ◽

Vol 20 (14) ◽

pp. 5343-5349 ◽

Cited By ~ 276

Author(s):

J. Cliff Yoon ◽

Troy W. Chickering ◽

Evan D. Rosen ◽

Barry Dussault ◽

Yubin Qin ◽

...

Keyword(s):

Transcriptional Activation ◽

Time Course ◽

Target Gene ◽

Target Genes ◽

Peroxisome Proliferator ◽

Peroxisome Proliferator Activated Receptor ◽

Isolation And Characterization ◽

Adipose Differentiation ◽

Novel Target

ABSTRACT The nuclear receptor peroxisome proliferator-activated receptor γ regulates adipose differentiation and systemic insulin signaling via ligand-dependent transcriptional activation of target genes. However, the identities of the biologically relevant target genes are largely unknown. Here we describe the isolation and characterization of a novel target gene induced by PPARγ ligands, termed PGAR (for PPARγ angiopoietin related), which encodes a novel member of the angiopoietin family of secreted proteins. The transcriptional induction of PGAR follows a rapid time course typical of immediate-early genes and occurs in the absence of protein synthesis. The expression of PGAR is predominantly localized to adipose tissues and placenta and is consistently elevated in genetic models of obesity. Hormone-dependent adipocyte differentiation coincides with a dramatic early induction of the PGAR transcript. Alterations in nutrition and leptin administration are found to modulate the PGAR expression in vivo. Taken together, these data suggest a possible role for PGAR in the regulation of systemic lipid metabolism or glucose homeostasis.

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β-Catenin–Histone Deacetylase Interactions Regulate the Transition of LEF1 from a Transcriptional Repressor to an Activator

Molecular and Cellular Biology ◽

10.1128/mcb.20.18.6882-6890.2000 ◽

2000 ◽

Vol 20 (18) ◽

pp. 6882-6890 ◽

Cited By ~ 158

Author(s):

Andrew N. Billin ◽

Hilary Thirlwell ◽

Donald E. Ayer

Keyword(s):

Enzymatic Activity ◽

Wnt Signaling ◽

Histone Deacetylase ◽

Promoter Region ◽

Target Gene ◽

Target Genes ◽

Transcriptional Repressor ◽

Downstream Target ◽

Step Mechanism

ABSTRACT Recent evidence suggests that certain LEF/TCF family members act as repressors in the absence of Wnt signaling. We show here that repression by LEF1 requires histone deacetylase (HDAC) activity. Further, LEF1 associates in vivo with HDAC1, and transcription of a model LEF1-dependent target gene is modulated by the ratio of HDAC1 to β-catenin, implying that repression by LEF1 is mediated by promoter-targeted HDAC. Consistent with this hypothesis, under repression conditions the promoter region of a LEF1 target gene is hypoacetylated. By contrast, when the reporter is activated, its promoter becomes hyperacetylated. Coexpression of β-catenin with LEF1 and HDAC1 results in the formation of a β-catenin/HDAC1 complex. Surprisingly, the enzymatic activity of HDAC1 associated with β-catenin is attenuated. Together, these findings imply that activation of LEF1-dependent genes by β-catenin involves a two-step mechanism. First, HDAC1 is dissociated from LEF1 and its enzymatic activity is attenuated. This first step yields a promoter that is inactive but poised for activation. Second, once HDAC1-dependent repression has been overridden, β-catenin binds LEF1 and the β-catenin–LEF1 complex is competent to activate the expression of downstream target genes.

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Differential transcriptional characteristics of small and large biliary epithelial cells derived from small and large bile ducts

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00237.2010 ◽

2010 ◽

Vol 299 (3) ◽

pp. G769-G777 ◽

Cited By ~ 16

Author(s):

S. Glaser ◽

M. Wang ◽

Y. Ueno ◽

J. Venter ◽

K. Wang ◽

...

Keyword(s):

Gene Expression ◽

Growth Hormone ◽

Epithelial Cells ◽

Target Gene ◽

Target Genes ◽

Biliary Epithelial Cells ◽

Gene Response ◽

Before And After ◽

In Vivo Growth

Biliary epithelial cells (BEC) are morphologically and functionally heterogeneous. To investigate the molecular mechanism for their diversities, we test the hypothesis that large and small BEC have disparity in their target gene response to their transcriptional regulator, the biliary cell-enriched hepatocyte nuclear factor HNF6. The expression of the major HNF ( HNF6, OC2, HNF1b, HNF1a, HNF4a, C/EBPb, and Foxa2) and representative biliary transport target genes that are HNF dependent were compared between SV40-transformed BEC derived from large (SV40LG) and small (SV40SM) ducts, before and after treatment with recombinant adenoviral vectors expressing HNF6 (AdHNF6) or control LacZ cDNA (AdLacZ). Large and small BEC were isolated from mouse liver treated with growth hormone, a known transcriptional activator of HNF6, and the effects on selected target genes were examined. Constitutive Foxa2, HNF1a, and HNF4a gene expression were 2.3-, 12.4-, and 2.6-fold, respectively, higher in SV40SM cells. This was associated with 2.7- and 4-fold higher baseline expression of HNF1a- and HNF4a-regulated ntcp and oatp1 genes, respectively. Following AdHNF6 infection, HNF6 gene expression was 1.4-fold higher ( P = 0.02) in AdHNF6 SV40SM relative to AdHNF6 SV40LG cells, with a corresponding higher Foxa2 (4-fold), HNF1a (15-fold), and HNF4a (6-fold) gene expression in AdHNF6-SV40SM over AdHNF6-SV40LG. The net effects were upregulation of HNF6 target gene glucokinase and of Foxa2, HNF1a, and HNF4a target genes oatp1, ntcp, and mrp2 over AdLacZ control in both cells, but with higher levels in AdH6-SV40SM over AdH6-SV40LG of glucokinase, oatp1, ntcp, and mrp2 (by 1.8-, 3.4-, 2.4-, and 2.5-fold, respectively). In vivo, growth hormone-mediated increase in HNF6 expression was associated with similar higher upregulation of glucokinase and mrp2 in cholangiocytes from small vs. large BEC. Small and large BEC have a distinct profile of hepatocyte transcription factor and cognate target gene expression, as well as differential strength of response to transcriptional regulation, thus providing a potential molecular basis for their divergent function.

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Ubiquitin specific peptidase Usp53 regulates osteoblast versus adipocyte lineage commitment

Scientific Reports ◽

10.1038/s41598-021-87608-x ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Hadla Hariri ◽

William N. Addison ◽

René St-Arnaud

Keyword(s):

Stromal Cells ◽

Target Gene ◽

Target Genes ◽

Nuclear Translocation ◽

Maturation Stage ◽

Mobility Shift ◽

Mobility Shift Assay ◽

Immunocompromised Mice ◽

The Impact

AbstractWe have previously shown that parathyroid hormone (PTH) induces the phosphorylation of the DNA-binding protein Nascent polypeptide associated complex And Coregulator alpha (NACA), leading to nuclear translocation of NACA and activation of target genes. Using ChIP-Seq against NACA in parallel with RNA-sequencing, we report the identification of Ubiquitin Specific Peptidase 53 (Usp53) as a target gene of PTH-activated NACA in osteoblasts. A binding site for NACA within the ChIP fragment from the Usp53 promoter was confirmed by electrophoretic mobility shift assay. Activity of the Usp53 promoter (− 2325/+ 238 bp) was regulated by the JUN-CREB complex and this activation relied on activated PKA and the presence of NACA. Usp53 knockdown in ST2 stromal cells stimulated expression of the osteoblastic markers Bglap2 (Osteocalcin) and Alpl (Alkaline phosphatase) and inhibited expression of the adipogenic markers Pparg and Cebpa. A similar effect was measured when knocking down Naca. During osteoblastogenesis, the impact of Usp53 knockdown on PTH responses varied depending on the maturation stage of the cells. In vivo implantation of Usp53-knockdown bone marrow stromal cells in immunocompromised mice showed an increase in osteoblast number and a decrease in adipocyte counts. Our data suggest that Usp53 modulates the fate of mesenchymal cells by impacting lineage selection.

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WT1 and DNMT3A Play an Essential Function and Represent Therapeutic Vulnerabilities in Certain AML Samples, As Shown By CRISPR/Cas9 Mediated Knockout in PDX Models In Vivo

Blood ◽

10.1182/blood-2021-150305 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 377-377

Author(s):

Maryam Ghalandary ◽

Yuqiao Gao ◽

Martin Becker ◽

Diana Amend ◽

Klaus H. Metzeler ◽

...

Keyword(s):

Flow Cytometry ◽

Cell Line ◽

Cell Lines ◽

Target Gene ◽

Target Genes ◽

Nsg Mice ◽

Essential Function ◽

Pdx Models

Abstract Background: The prognosis of patients with acute myeloid leukemia (AML) remains poor and novel therapeutic options are intensively needed. Targeted therapies specifically address molecules with essential function for AML and deciphering novel essential target genes is of utmost importance. Functional genomics via CRISPR\Cas9 technology paves the way for the systematic discovery of novel essential genes, but was so far mostly restricted to studying cell lines in vitro, lacking features of, e.g., primary tumor cells and the in vivo tumor microenvironment. To move closer to the clinical situation in patients, we used the CRISPR\Cas9 technology in patient-derived xenograft (PDX) models of AML in vivo. Methods: Primary tumor cells from seven patients with AML were transplanted into immunocompromised NSG mice and serially transplantable PDX models derived thereof. PDX models were selected which carry the AML specific mutations of interest at variant allele frequencies close to 0.5. PDX cells were lentivirally transduced to express the Cas9 protein and a sgRNA; successfully transduced PDX cells were enriched by flow cytometry gating on a recombinant fluorochrome or by puromycin. The customized sgRNA library was designed using the CLUE (www.crispr-clue.de) platform and cloned into a lentiviral vector with five different sgRNAs per target gene, plus positive and negative controls (Becker et al., Nucleic Acids Res. 2020). PDX cells were lentivirally transduced with the CRISPR/Cas9 sgRNA library, transplanted into NSG mice, grown in vivo and cells re-isolated at advanced AML disease. sgRNA distribution was measured by next generation sequencing and compared to input control using the MAGeCK pipeline. Interesting dropout hits from PDX in vivo screens were validated by fluorochrome-guided competitive in vivo experiments in the PDX models, comparing growth of PDX AML cells with knockout of the gene of interest versus control knockout in the same mouse. PDX cells were transduced with lentiviral vectors expressing a single sgRNA, using in parallel three different sgRNAs per target gene. Targeting and control sgRNAs were marked by different fluorochromes; PDX cells expressing targeting or control sgRNA were mixed at a 1:1 ratio, injected into NSG mice and PDX models competitively grown until advanced disease stage, when cell distributions was determined by flow cytometry. Human AML cell lines were studied in vitro for comparison. Results: In search for genes with essential function in AML, we cloned a small customized sgRNA library targeting 34 genes recurrently mutated in AML and tested the library in two PDX AML models in vivo. From the dropouts, we validated most interesting target genes using fluorochrome-guided competitive in vivo assays. Knockout of NPM1 abrogated in vivo growth in all PDX AML models tested, reproducing the known common essential function of NPM1. KRAS proved an essential function in PDX AML models both with and without an oncogenic mutation in KRAS, although with a stronger effect upon KRAS mutation, suggesting that patients with tumors both with and without KRAS mutation might benefit from treatment inhibiting KRAS. Surprising results were obtained for WT1 and DNMT3A. Both genes are frequently mutated in AML, but most AML cell lines tested in vitro do not show an essential function of any of the two genes, in published knockdown or knockout data, including from the Cancer Dependency Map database. On the contrary, knockout of either WT1 or DNMT3A was shown to enhance growth of AML cell lines and increase leukemogenesis in certain models. In PDX models in vivo, we found a clearly essential function for DNMT3A in all AML samples and WT1 in most samples tested and PDX in vivo results were discordant to cell line in vitro data, suggesting that cell line inherent features and/or the in vivo environment influence the function of WT1 and DNMT3A. Conclusion: We conclude that functional genomics in PDX models in vivo allows discovering essentialities hidden for cell line in vitro approaches. WT1 and DNMT3A harbor the potential to represent attractive therapeutic targets in AML under in vivo conditions, warranting further evaluation. Disclosures No relevant conflicts of interest to declare.

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Mediator complex subunit Med19 binds directly GATA DNA-binding zinc finger and functions with Med1 in GATA-driven gene regulation in vivo

10.1101/2020.04.03.023895 ◽

2020 ◽

Author(s):

Clément Immarigeon ◽

Sandra Bernat-Fabre ◽

Emmanuelle Guillou ◽

Alexis Verger ◽

Elodie Prince ◽

...

Keyword(s):

Dna Binding ◽

Rna Polymerase Ii ◽

Zinc Finger ◽

Target Gene ◽

Target Genes ◽

Mediator Complex ◽

Transcriptional Responses ◽

Evolutionarily Conserved

AbstractThe evolutionarily-conserved multiprotein Mediator complex (MED) serves as an interface between DNA-bound transcription factors (TFs) and the RNA Polymerase II machinery. It has been proposed that each TF interacts with a dedicated MED subunit to induce specific transcriptional responses. However, binary MED subunit - TF partnerships are probably oversimplified models. Using Drosophila TFs of the GATA family - Pannier (Pnr) and Serpent (Srp) - as a model, we have previously established GATA cofactor evolutionarily-conserved function for the Med1 Mediator subunit. Here, we show that another subunit, Med19, is required for GATA-dependent gene expression and interacts physically with Pnr and Srp in cellulo, in vivo and in vitro through their conserved C-zinc finger (ZF), indicating general GATA co-activator functions. Interestingly, Med19 is critical for the regulation of all tested GATA target genes which is not the case for Med1, suggesting differential use of MED subunits by GATAs depending on the target gene. Lastly, despite their presumed distant position within the MED middle module, both subunits interact physically. In conclusion, our data shed new light first on the MED complex, engaging several subunits to mediate TF-driven transcriptional responses and second, on GATA TFs, showing that ZF DNA-binding domain also serves for transactivation.

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