scholarly journals Gene dosage effects of polyA track engineered hypomorphs

2021 ◽  
Author(s):  
Geralle N. Powell ◽  
Slavica Pavlovic-Djuranovic ◽  
Sergej Djuranovic
Keyword(s):  
Target Genes ◽  
Gene Dosage ◽  
Functional Characterization ◽  
Interleukin 2 ◽  
Regulatory Sequences ◽  
Mrna Levels ◽  
Hypomorphic Mutation ◽  
Downstream Target ◽  
Gene Dosage Effects ◽  
The Creation

ABSTRACTThe manipulation of gene activity through the creation of hypomorphic mutants has been a long-standing tool in examining gene function. Our previous studies have indicated that hypomorphic mutants could be created through the insertion of cis-regulatory sequences composed of consecutive adenosine nucleotides called polyA tracks. Here we confirm that this method can be used for the creation of hypomorphic mutants and functional characterization of membrane, secretory and endogenous proteins. Insertion of polyA tracks into the sequences of interleukin-2 and membrane protein CD20 results in a programmable reduction of mRNA stability and attenuation of protein expression regardless of the presence of signaling sequence. Likewise, CRISPR/Cas9 targeted insertion of polyA tracks in the coding sequence of endogenous human genes AUF1 and TP53 results in a programmable reduction of targeted protein and mRNA levels. Functional analyses of AUF1 engineered hypomorphs indicate a direct correlation between AUF1 gene levels and the stability of AUF1-regulated mRNAs. Hypomorphs of TP53 affect the expression of the downstream target genes differentially depending upon the severity of the hypomorphic mutation. Finally, decreases in TP53 protein affect the same cellular pathways in polyA track engineered cells as in cancer cells, indicating these variants’ biological relevance. These results highlight this technology’s power to create predictable, stable hypomorphs in recombinant or endogenous genes in combination with CRISPR/Cas9 engineering tools.

scholarly journals Icariin Intervenes in Cardiac Inflammaging through Upregulation of SIRT6 Enzyme Activity and Inhibition of the NF-Kappa B Pathway

2015 ◽  
Vol 2015 ◽  
pp. 1-12 ◽  
Author(s):  
Yang Chen ◽  
Tao Sun ◽  
Junzhen Wu ◽  
Bill Kalionis ◽  
Changcheng Zhang ◽  
...  
Keyword(s):  
Protein Expression ◽  
Enzymatic Activity ◽  
Histone Deacetylase ◽  
Target Genes ◽  
Inhibitory Effect ◽  
Mrna Levels ◽  
Cell Models ◽  
Animal Tissues ◽  
Nf Kappa B ◽  
Downstream Target

The aim of the study was to investigate the effect of icariin (ICA) on cardiac aging through its effects on the SIRT6 enzyme and on the NF-κB pathway. Investigating the effect of ICA on the enzymatic activity of histone deacetylase SIRT6 revealed a concentration of 10−8 mol/L ICA had a maximum activating effect on histone deacetylase SIRT6 enzymatic activity. Western analysis showed that ICA upregulated SIRT6 protein expression and downregulated NF-κB (p65) protein expression in animal tissues and cell models. ICA upregulated the expression of SIRT6 and had an inhibitory effect on NF-κB inflammatory signaling pathways as shown by decreasing mRNA levels of the NF-κB downstream target genes TNF-α, ICAM-1, IL-2, and IL-6. Those effects were mediated directly or indirectly by SIRT6. We provided evidence that inflammaging may involve a novel link between the effects of ICA on SIRT6 (a regulator of aging) and NF-κB (a regulator of inflammation).

p53 protein and p21 mRNA levels and caspase-3 activity are altered by zinc status in aortic endothelial cells

2002 ◽  
Vol 283 (2) ◽  
pp. C631-C638 ◽  
Author(s):  
Jessica C. Fanzo ◽  
Scott K. Reaves ◽  
Libin Cui ◽  
Lei Zhu ◽  
Kai Y. Lei
Keyword(s):  
Endothelial Cells ◽  
Caspase 3 ◽  
Target Genes ◽  
P53 Protein ◽  
Mrna Abundance ◽  
Mrna Levels ◽  
Zinc Status ◽  
Aortic Endothelial Cells ◽  
Downstream Target ◽  
Aortic Endothelial

The influence of zinc status on the levels of p53, as well as downstream targets of p53 in cell repair and survival, was examined in human aortic endothelial cells (HAECs). A serum-reduced low-zinc medium (ZD) was used to deplete zinc over one passage. Other treatments included zinc-normal control (ZN), zinc-adequate (ZA), and zinc-supplemented (ZS) treatment with 3.0, 16.0, and 32.0 μM zinc, respectively. Cellular zinc levels in the ZD cells were 64% of ZN controls; levels in the ZA cells were not different, but levels in ZS cells were significantly higher (40%) than in ZN cells. No difference in p53 mRNA abundance was detected among all treatments; however, p53 nuclear protein levels were >100% higher in the ZD and ZS cells and almost 200% higher in the ZA cells than in ZN controls. In addition, p21 mRNA abundance, a downstream target of p53 protein, was increased in the ZS cells compared with both the ZN control and ZD cells. In the ZS cells, bax and mcl-1 were also ∼50% higher compared with ZN controls, whereas bcl-2 mRNA was increased compared with ZA cells. Moreover, caspase-3 activity of ZD cells was not different from that of ZN controls but was reduced to 83 and 69% of ZN controls in ZA and ZS cells, respectively. Thus p53 protein and p53 downstream target genes appeared to be modulated by intracellular zinc status in HAECs.

scholarly journals Increased circulating miR-370-3p regulates steroidogenic factor 1 in endometriosis

2019 ◽  
Vol 316 (3) ◽  
pp. E373-E382 ◽  
Author(s):  
Zhuoying Hu ◽  
Ramanaiah Mamillapalli ◽  
Hugh S. Taylor
Keyword(s):  
Cell Proliferation ◽  
Target Genes ◽  
Regulatory Protein ◽  
Negative Regulator ◽  
Mrna Levels ◽  
Steroidogenic Factor 1 ◽  
Altered Expression ◽  
Downstream Target ◽  
Steroidogenic Acute Regulatory ◽  
Induced Apoptosis

Endometriosis is a gynecologic disease common among reproductive-aged women caused by the growth of endometrial tissue outside the uterus. Altered expression of numerous genes and microRNAs has been reported in endometriosis. Steroidogenic factor 1 (SF-1), an essential transcriptional regulator of multiple genes involved in estrogen biosynthesis, is aberrantly increased and plays an important role in the pathogenesis of endometriosis. Here, we show the expression of SF-1 in endometriosis is regulated by miR-370-3p. Sera and tissue were collected from 20 women surgically diagnosed with endometriosis and 26 women without endometriosis. We found that miR-370-3p levels were decreased in the serum of patients with endometriosis while SF-1 mRNA levels were inversely upregulated in endometriotic lesions compared with respective controls. Transfection of primary endometriotic cells with miR-370-3p mimic or inhibitor resulted in the altered expression of SF-1 and SF-1 downstream target genes steroidogenic acute regulatory protein (StAR) and CYP19A1. Overexpression of miR-370-3p inhibited cell proliferation and induced apoptosis in endometriotic cells. This study reveals that miR-370-3p functions as a negative regulator of SF-1 and cell proliferation in endometriotic cells. We suggest a novel therapeutic strategy for controlling SF-1 in endometriosis.

scholarly journals Defining Essential Enhancer for Pluripotent stem cells using Features Oriented CRISPR-Cas9 Screen

2019 ◽  
Author(s):  
Hao Fei Wang ◽  
Tushar Warrier ◽  
Chadi EL Farran ◽  
Zheng Zihao ◽  
Qiao Rui Xing ◽  
...  
Keyword(s):  
Cell Fate ◽  
Target Genes ◽  
Es Cells ◽  
Functional Characterization ◽  
Regulatory Elements ◽  
Downstream Target ◽  
Mouse Es Cells ◽  
Cellular Processes ◽  
Stat3 Signaling ◽  
Stat3 Signaling Pathway

ABSTRACTCis Regulatory Elements (CREs) regulate the expression of the genes in their genomic neighborhoods and influence cellular processes such as cell-fate maintenance and differentiation. To date, there remain major gaps in the functional characterization of CREs and the identification of its target genes in the cellular native environment. In this study, we performed a Features Oriented CRISPR Utilized Systematic (FOCUS) screen of OCT4-bound CREs using CRISPR/Cas9 to identify functional enhancers important for pluripotency maintenance in mouse ES cells. From the initial 235 candidates tested, 16 CREs were identified to be essential stem cell enhancers. Using RNA-seq and genomic 4C-seq, we further uncovered a complex network of candidate CREs and their downstream target genes, which supports the growth and self-renewal of mESCs. Notably, an essential enhancer, CRE111, and its target, Lrrc31, form the important switch to modulate the LIF-JAK1-STAT3 signaling pathway.

scholarly journals Mouse Homologue of the Schizophrenia Susceptibility GeneZNF804Aas a Target of Hoxc8

2010 ◽  
Vol 2010 ◽  
pp. 1-7 ◽  
Author(s):  
Hyun Joo Chung ◽  
Ji-Yeon Lee ◽  
Custer C. Deocaris ◽  
Hyehyun Min ◽  
Sang Hoon Kim ◽  
...  
Keyword(s):  
Complex Traits ◽  
Target Genes ◽  
Zinc Finger Protein ◽  
Mouse Embryos ◽  
Regulatory Control ◽  
Mrna Levels ◽  
F9 Cells ◽  
Downstream Target ◽  
Cloning Technique

Using a ChIP-cloning technique, we identified a Zinc finger protein 804a (Zfp804a) as one of the putative Hoxc8 downstream target genes. We confirmed binding of Hoxc8 to an intronic region ofZfp804aby ChIP-PCR in F9 cells as well as in mouse embryos. Hoxc8 upregulatedZfp804amRNA levels and augmented minimal promoter activity in vitro. In E11.5 mouse embryos,Zfp804aandHoxc8were coexpressed. Recent genome-wide studies identifiedZfp804a(orZNF804Ain humans) as a plausible marker for schizophrenia, leading us to hypothesize that this embryogenic regulatory control might also exert influence in development of complex traits such as psychosis.

scholarly journals OMIC-03. TRANSLATIONAL CONTROL IN MYC AND MYCN MEDULLOBLASTOMA

2021 ◽  
Vol 23 (Supplement_1) ◽  
pp. i37-i37
Author(s):  
Christin Schmidt ◽  
Albertas Navickas ◽  
Frederique Zindy ◽  
Dana Farmer ◽  
Davide Ruggero ◽  
...  
Keyword(s):  
Translational Control ◽  
Target Genes ◽  
Xenograft Model ◽  
Precursor Cells ◽  
Neural Precursor Cells ◽  
Regulatory Function ◽  
Neural Precursor ◽  
Transcriptional Level ◽  
Regulatory Sequences ◽  
Mrna Levels

Abstract Medulloblastoma has been extensively characterized at the genomic and transcriptional levels, but little is known about how alterations in translational control underlie tumor development. Myc and Mycn are often deregulated in medulloblastoma and play important roles in tumor initiation, maintenance and progression. Although both proteins have similar structures and are functionally redundant in hindbrain development, their amplification in cerebellar granule neural precursor cells leads to different medulloblastoma subtypes. In this project we are employing ribosome profiling on mouse medulloblastoma tumors generated from granule neural precursor cells with enforced expression of Myc or Mycn. Ribosome-protected mRNA sequencing allows us to quantitatively assess the specific transcripts regulated at the level of translation, identify translation regulatory sequences within the mammalian transcriptome, and understand genotype-to-phenotype processes. We discovered that Myc- and Mycn-driven tumors exhibit many more changes at the translational rather than at the transcriptional level. In particular, we found that Mycn-driven medulloblastoma upregulates the translation of Myc target genes, while mRNA levels of those genes show no difference between Myc- and Mycn-driven tumors. Furthermore, we find that the most significant translationally upregulated Myc target genes in the Mycn tumors are transcripts that encode ribosome biogenesis factors. We will further study the role of Myc and Mycn on translational regulation of the medulloblastoma transcriptome using our xenograft model of human iPSC-derived neuroepithelial stem cells overexpressing Myc or Mycn. Our goal is to understand the regulatory function of the translational landscape in Myc- and Mycn-driven medulloblastoma and to decipher the oncogenic signaling cascades leading to different medulloblastoma subtypes.

scholarly journals Regulatory Network Analysis in Estradiol-Treated Human Endothelial Cells

2021 ◽  
Vol 22 (15) ◽  
pp. 8193
Author(s):  
Daniel Pérez-Cremades ◽  
Ana B. Paes ◽  
Xavier Vidal-Gómez ◽  
Ana Mompeón ◽  
Carlos Hermenegildo ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Transcription Factor ◽  
Transcription Factors ◽  
Regulatory Network ◽  
Mirna Target ◽  
Target Genes ◽  
Functional Characterization ◽  
Human Endothelial Cells ◽  
Mrna Targets ◽  
Canonical Pathways

Background/Aims: Estrogen has been reported to have beneficial effects on vascular biology through direct actions on endothelium. Together with transcription factors, miRNAs are the major drivers of gene expression and signaling networks. The objective of this study was to identify a comprehensive regulatory network (miRNA-transcription factor-downstream genes) that controls the transcriptomic changes observed in endothelial cells exposed to estradiol. Methods: miRNA/mRNA interactions were assembled using our previous microarray data of human umbilical vein endothelial cells (HUVEC) treated with 17β-estradiol (E2) (1 nmol/L, 24 h). miRNA–mRNA pairings and their associated canonical pathways were determined using Ingenuity Pathway Analysis software. Transcription factors were identified among the miRNA-regulated genes. Transcription factor downstream target genes were predicted by consensus transcription factor binding sites in the promoter region of E2-regulated genes by using JASPAR and TRANSFAC tools in Enrichr software. Results: miRNA–target pairings were filtered by using differentially expressed miRNAs and mRNAs characterized by a regulatory relationship according to miRNA target prediction databases. The analysis identified 588 miRNA–target interactions between 102 miRNAs and 588 targets. Specifically, 63 upregulated miRNAs interacted with 295 downregulated targets, while 39 downregulated miRNAs were paired with 293 upregulated mRNA targets. Functional characterization of miRNA/mRNA association analysis highlighted hypoxia signaling, integrin, ephrin receptor signaling and regulation of actin-based motility by Rho among the canonical pathways regulated by E2 in HUVEC. Transcription factors and downstream genes analysis revealed eight networks, including those mediated by JUN and REPIN1, which are associated with cadherin binding and cell adhesion molecule binding pathways. Conclusion: This study identifies regulatory networks obtained by integrative microarray analysis and provides additional insights into the way estradiol could regulate endothelial function in human endothelial cells.

scholarly journals Extract of Wax Gourd Peel Prevents High-Fat Diet-Induced Hyperlipidemia in C57BL/6 Mice via the Inhibition of the PPARγPathway

2013 ◽  
Vol 2013 ◽  
pp. 1-11 ◽  
Author(s):  
Ming Gu ◽  
Shengjie Fan ◽  
Gaigai Liu ◽  
Lu Guo ◽  
Xiaobo Ding ◽  
...  
Keyword(s):  
Blood Glucose ◽  
High Fat Diet ◽  
Target Genes ◽  
Metabolic Diseases ◽  
Body Weight Gain ◽  
Fasting Blood Glucose ◽  
Peroxisome Proliferator ◽  
Mrna Levels ◽  
High Fat ◽  
Wax Gourd

Wax gourd is a popular vegetable in East Asia. In traditional Chinese medicine, wax gourd peel is used to prevent and treat metabolic diseases such as hyperlipidemia, hyperglycemia, obesity, and cardiovascular disease. However, there is no experimental evidence to support these applications. Here, we examined the effect of the extract of wax gourd peel (EWGP) on metabolic disorders in diet-induced C57BL/6 obese mice. In the preventive experiment, EWGP blocked body weight gain and lowered serum total cholesterol (TC), low-density lipoprotein cholesterol (LDL-c), liver TG and TC contents, and fasting blood glucose in mice fed with a high-fat diet. In the therapeutic study, we induced obesity in the mice and treated with EWGP for two weeks. We found that EWGP treatment reduced serum and liver triglyceride (TG) contents and fasting blood glucose and improved glucose tolerance in the mice. Reporter assay and gene expression analysis showed that EWGP could inhibit peroxisome proliferator-activated receptorγ(PPARγ) transactivities and could decrease mRNA levels of PPARγand its target genes. We also found that HMG-CoA reductase (HMGCR) was downregulated in the mouse liver by EWGP. Our data suggest that EWGP lowers hyperlipidemia of C57BL/6 mice induced by high-fat diet via the inhibition of PPARγand HMGCR signaling.

scholarly journals In Silico Identification and Functional Characterization of Conserved miRNAs in the Genome of Cryptosporidium parvum

2021 ◽  
Vol 15 ◽  
pp. 117793222110276
Author(s):  
Md. Irtija Ahsan ◽  
Md. Shahidur Rahman Chowdhury ◽  
Moumita Das ◽  
Sharmin Akter ◽  
Sawrab Roy ◽  
...  
Keyword(s):  
Cryptosporidium Parvum ◽  
Target Genes ◽  
Expressed Sequence Tag ◽  
Functional Characterization ◽  
Childhood Mortality ◽  
Homology Search ◽  
Health Concern ◽  
Nutrient Metabolism ◽  
Screening Process ◽  
Computational Screening

Cryptosporidium parvum, a predominant causal agent of a fatal zoonotic protozoan diarrhoeal disease called cryptosporidiosis, bears a worldwide public health concern for childhood mortality and poses a key threat to the dairy and water industries. MicroRNAs (miRNAs), small but powerful posttranscriptional gene silencing RNA molecules, regulate a variety of molecular, biological, and cellular processes in animals and plants. As to the present date, there is a paucity of information regarding miRNAs of C. parvum; hence, this study was used to identify miRNAs in the organism using a comprehensible expressed sequence tag–based homology search approach consisting of a series of computational screening process from the identification of putative miRNA candidates to the functional annotation of the important gene targets in C. parvum. The results revealed a conserved miRNA that targeted 487 genes in the model organism ( Drosophila melanogaster) and 85 genes in C. parvum, of which 11 genes had direct involvements in several crucial virulence factors such as environmental oocyst protection, excystation, locomotion, adhesion, invasion, stress protection, intracellular growth, and survival. Besides, 20 genes showed their association with various major pathways dedicated for the ribosomal biosynthesis, DNA repair, transportation, protein production, gene expression, cell cycle, cell proliferation, development, immune response, differentiation, and nutrient metabolism of the organism in the host. Thus, this study provides a strong evidence of great impact of identified miRNA on the biology, virulence, and pathogenesis of C. parvum. Furthermore, the study suggests that the detected miRNA could be a potential epigenomic tool for controlling the protozoon through silencing those virulent and pathway-related target genes.

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