scholarly journals Poly(ADP-ribosyl)ation associated changes in CTCF-chromatin binding and gene expression in breast cells

2017 ◽  
Author(s):  
Ioanna Pavlaki ◽  
France Docquier ◽  
Igor Chernukhin ◽  
Georgia Kita ◽  
Svetlana Gretton ◽  
...  
Keyword(s):  
Gene Expression ◽  
Molecular Mass ◽  
Molecular Mechanisms ◽  
Apparent Molecular Mass ◽  
Ctcf Binding ◽  
Binding Motif ◽  
Cellular Functions ◽  
Altered Expression ◽  
Cycle Arrest ◽  
Breast Cells

AbstractCTCF is an evolutionarily conserved and ubiquitously expressed architectural protein regulating a plethora of cellular functions via different molecular mechanisms. CTCF can undergo a number of post-translational modifications which change its properties and functions. One such modifications linked to cancer is poly(ADP-ribosyl)ation (PARylation). The highly PARylated CTCF form has an apparent molecular mass of 180 kDa (referred to as CTCF180), which can be distinguished from hypo- and non-PARylated CTCF with the apparent molecular mass of 130 kDa (referred to as CTCF130). The existing data accumulated so far have been mainly related to CTCF130. However, the properties of CTCF180 are not well understood despite its abundance in a number of primary tissues. In this study we performed ChIP-seq and RNA-seq analyses in human breast cells 226LDM, which display predominantly CTCF130 when proliferating, but CTCF180 upon cell cycle arrest. We observed that in the arrested cells the majority of sites lost CTCF, whereas fewer sites gained CTCF or remain bound (i.e. common sites). The classical CTCF binding motif was found in the lost and common, but not in the gained sites. The changes in CTCF occupancies in the lost and common sites were associated with increased chromatin densities and altered expression from the neighboring genes. Based on these results we propose a model integrating the CTCF130/180 transition with CTCF-DNA binding and gene expression changes. This study also issues an important cautionary note concerning the design and interpretation of any experiments using cells and tissues where CTCF180 may be present.

scholarly journals Genome-scale mapping reveals complex regulatory activities of RpoN in Yersinia pseudotuberculosis

2020 ◽  
Author(s):  
F.A.K.M. Mahmud ◽  
K. Nilsson ◽  
A. Fahlgren ◽  
R. Navais ◽  
R. Choudhry ◽  
...  
Keyword(s):  
Gene Expression ◽  
Binding Sites ◽  
Sigma Factor ◽  
Biofilm Formation ◽  
Yersinia Pseudotuberculosis ◽  
Regulation Of Gene Expression ◽  
Binding Motif ◽  
Alternative Sigma Factor ◽  
Altered Expression ◽  
Sense Strand

ABSTRACTRpoN, an alternative sigma factor commonly known as sigma 54, is implicated in persistent stages of Yersinia pseudotuberculosis infections in which genes associated with this regulator are upregulated. We here combined phenotypic and genomic assays to provide insight into its role and function in this pathogen. RpoN was found essential for Y. pseudotuberculosis virulence in mice, and in vitro functional assays showed that it controls biofilm formation and motility. Mapping genome-wide associations of Y. pseudotuberculosis RpoN using chromatin immunoprecipitation coupled with next-generation sequencing identified an RpoN-binding motif located at 103 inter- and intragenic sites on both sense and anti-sense strands. Deletion of rpoN had a large impact on gene expression, including down-regulation of genes encoding proteins involved in flagellar assembly, chemotaxis, and quorum sensing. There were also clear indications of cross talk with other sigma factors, together with indirect effects due to altered expression of other regulators. Matching differential gene expression with locations of the binding sites implicated around 130 genes or operons potentially activated or repressed by RpoN. Mutagenesis of selected intergenic binding sites confirmed both positive and negative regulatory effects of RpoN binding. Corresponding mutations of intragenic sense sites had less impact on associated gene expression. Surprisingly, mutating intragenic sites on the anti-sense strand commonly reduced expression of genes encoded by the corresponding sense strand.IMPORTANCEThe alternative sigma factor, RpoN (σ 54), which is widely distributed in eubacteria have been implicated to control gene expression of importance for numerous functions including virulence. Proper responses to host environments are crucial for bacteria to establish infection and regulatory mechanisms involved are therefore of high interest for development of future therapeutics. Little is known about the function of RpoN in the intestinal pathogen Y. pseudotuberculosis and we therefore investigated its regulatory role in this pathogen. This regulator was indeed found to be critical for establishment of infection in mice, likely involving its requirement for motility and biofilm formation. The RpoN regulon involved both activating and suppressive effects on gene expression which could be confirmed with mutagenesis of identified binding sites. This is the first of its kind study of RpoN in Y. pseudotuberculosis revealing complex regulation of gene expression involving both productive and silent effects of its binding to DNA providing important information about RpoN regulation in enterobacteria.

scholarly journals Gene expression levels of DNA methyltransferase enzymes in Shank3-deficient mouse model of autism during early development

2021 ◽  
Vol 55 (4) ◽  
pp. 234-237
Author(s):  
Annamaria Srancikova ◽  
Alexandra Reichova ◽  
Zuzana Bacova ◽  
Jan Bakos
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Brain Development ◽  
Early Development ◽  
Molecular Mechanisms ◽  
Altered Expression ◽  
Expression Levels ◽  
The Brain ◽  
Deficient Mice ◽  
Gene Expression Levels

Abstract Objectives. The balance between DNA methylation and demethylation is crucial for the brain development. Therefore, alterations in the expression of enzymes controlling DNA methylation patterns may contribute to the etiology of neurodevelopmental disorders, including autism. SH3 and multiple ankyrin repeat domains 3 (Shank3)-deficient mice are commonly used as a well-characterized transgenic model to investigate the molecular mechanisms of autistic symptoms. DNA methyltransferases (DNMTs), which modulate several cellular processes in neurodevelopment, are implicated in the pathophysiology of autism. In this study, we aimed to describe the gene expression changes of major Dnmts in the brain of Shank3-deficient mice during early development. Methods and Results. The Dnmts gene expression was analyzed by qPCR in 5-day-old homo-zygous Shank3-deficient mice. We found significantly lower Dnmt1 and Dnmt3b gene expression levels in the frontal cortex. However, no such changes were observed in the hippocampus. However, significant increase was observed in the expression of Dnmt3a and Dnmt3b genes in the hypothalamus of Shank3-deficient mice. Conclusions. The present data indicate that abnormalities in the Shank3 gene are accompanied by an altered expression of DNA methylation enzymes in the early brain development stages, therefore, specific epigenetic control mechanisms in autism-relevant models should be more extensively investigated.

scholarly journals In utero Bisphenol A Exposure Is Linked with Sex Specific Changes in the Transcriptome and Methylome of Human Amniocytes

2019 ◽  
Vol 105 (2) ◽  
pp. 453-467
Author(s):  
Amita Bansal ◽  
Nicole Robles-Matos ◽  
Paul Zhiping Wang ◽  
David E Condon ◽  
Apoorva Joshi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Bisphenol A ◽  
Gestational Age ◽  
Molecular Mechanisms ◽  
In Utero ◽  
Metabolic Dysfunction ◽  
Altered Expression ◽  
Obesity And Diabetes ◽  
Genome Wide

Abstract Context Prenatal exposure to bisphenol A (BPA) is linked to obesity and diabetes but the molecular mechanisms driving these phenomena are not known. Alterations in deoxyribonucleic acid (DNA) methylation in amniocytes exposed to BPA in utero represent a potential mechanism leading to metabolic dysfunction later in life. Objective To profile changes in genome-wide DNA methylation and expression in second trimester human amniocytes exposed to BPA in utero. Design A nested case-control study was performed in amniocytes matched for offspring sex, maternal race/ethnicity, maternal age, gestational age at amniocentesis, and gestational age at birth. Cases had amniotic fluid BPA measuring 0.251 to 23.74 ng/mL. Sex-specific genome-wide DNA methylation analysis and RNA-sequencing (RNA-seq) were performed to determine differentially methylated regions (DMRs) and gene expression changes associated with BPA exposure. Ingenuity pathway analysis was performed to identify biologically relevant pathways enriched after BPA exposure. In silico Hi-C analysis identified potential chromatin interactions with DMRs. Results There were 101 genes with altered expression in male amniocytes exposed to BPA (q < 0.05) in utero, with enrichment of pathways critical to hepatic dysfunction, collagen signaling and adipogenesis. Thirty-six DMRs were identified in male BPA-exposed amniocytes and 14 in female amniocyte analysis (q < 0.05). Hi-C analysis identified interactions between DMRs and 24 genes with expression changes in male amniocytes and 12 in female amniocytes (P < 0.05). Conclusion In a unique repository of human amniocytes exposed to BPA in utero, sex-specific analyses identified gene expression changes in pathways associated with metabolic disease and novel DMRs with potential distal regulatory functions.

scholarly journals <i>In Vitro</i> Exposure to Isoprene-Derived Secondary Organic Aerosol by Direct Deposition and its Effects on <i>COX-2</i> and <i>IL-8</i> Gene Expression

2016 ◽  
Author(s):  
Maiko Arashiro ◽  
Ying-Hsuan Lin ◽  
Kenneth G. Sexton ◽  
Zhenfa Zhang ◽  
Ilona Jaspers ◽  
...  
Keyword(s):  
Gene Expression ◽  
Molecular Mechanisms ◽  
Secondary Organic Aerosol ◽  
Organic Aerosol ◽  
Mrna Levels ◽  
Altered Expression ◽  
Direct Deposition ◽  
Discovery Research ◽  
Human Lung Cells ◽  
Cox 2

Abstract. Atmospheric oxidation of isoprene, the most abundant non-methane hydrocarbon emitted into Earth’s atmosphere primarily from terrestrial vegetation, is now recognized as a major contributor to the global secondary organic aerosol (SOA) burden. Anthropogenic pollutants significantly enhance isoprene SOA formation through acid-catalyzed heterogeneous chemistry of epoxide products. Since isoprene SOA formation as a source of fine aerosol is a relatively recent discovery, research is lacking on evaluating its potential adverse effects on human health. The objective of this study was to examine the effect of isoprene-derived SOA on inflammation-associated gene expression in human lung cells using a direct deposition exposure method. We assessed altered expression of inflammation-related genes in human bronchial epithelial cells (BEAS-2B) exposed to isoprene-derived SOA generated in an outdoor chamber facility. Measurements of gene expression of known inflammatory biomarkers interleukin 8 (IL-8) and cyclooxygenase 2 (COX-2) in exposed cells, together with complementary chemical measurements, showed that a dose of 0.067 µg cm−2 of SOA from isoprene photooxidation leads to statistically significant increases in IL-8 and COX-2 mRNA levels. Resuspension exposures using aerosol filter extracts corroborated these findings, supporting the conclusion that isoprene-derived SOA constituents induce the observed changes in mRNA levels. Future studies are needed to systematically examine the molecular mechanisms of toxicity.

scholarly journals Affinity for the nuclear compartment and expression during cell differentiation implicate phosphorylated Groucho/TLE1 forms of higher molecular mass in nuclear functions

1996 ◽  
Vol 317 (2) ◽  
pp. 523-531 ◽  
Author(s):  
Junaid HUSAIN ◽  
Rita LO ◽  
Diane GRBAVEC ◽  
Stefano STIFANI
Keyword(s):  
Molecular Mass ◽  
Neural Development ◽  
Molecular Mechanisms ◽  
Signal Transduction Pathway ◽  
Apparent Molecular Mass ◽  
Nuclear Compartment ◽  
Drosophila Protein ◽  
Molecular Masses ◽  
Redundant Functions ◽  
Embryonic Segmentation

The Drosophila protein Groucho is involved in embryonic segmentation and neural development, and is implicated in the Notch signal transduction pathway. We are investigating the molecular mechanisms underlying the function of Groucho and of its mammalian homologues, the TLE (‘transducin-like Enhancer of split’) proteins. We show that Groucho/TLE1 proteins are phosphorylated. We also show that two populations of phosphorylated Groucho proteins can be identified based on their interaction with the nuclear compartment. More slowly migrating proteins with an apparent molecular mass of roughly 110 kDa interact strongly with the nuclei, while faster migrating proteins displaying molecular masses of roughly 84–85 kDa show lower affinity for the nuclear compartment. Similarly, TLE1 proteins with an apparent molecular mass of roughly 118 kDa exhibit higher affinity for the nuclear compartment than do faster migrating forms with apparent molecular masses of 90–93 kDa. Moreover, we show that the nuclear, more slowly migrating, TLE1 proteins are induced during neural determination of P19 embryonic carcinoma cells. These results implicate phosphorylation in the activity of Groucho/TLE1 proteins and suggest that phosphorylated forms of higher molecular mass are involved in nuclear functions. Finally, we show that different TLE proteins respond in different ways to the neural commitment of P19 cells, suggesting that individual members of this protein family may have non-redundant functions.

scholarly journals Activation of human heat shock genes is accompanied by oligomerization, modification, and rapid translocation of heat shock transcription factor HSF1.

1993 ◽  
Vol 13 (4) ◽  
pp. 2486-2496 ◽  
Author(s):  
R Baler ◽  
G Dahl ◽  
R Voellmy
Keyword(s):  
Gene Expression ◽  
Heat Treatment ◽  
Transcription Factor ◽  
Heat Shock ◽  
Dna Binding ◽  
Molecular Mass ◽  
Polyclonal Antibodies ◽  
Protein S ◽  
Apparent Molecular Mass ◽  
Nuclear Fraction

Transcriptional activity of heat shock (hsp) genes is controlled by a heat-activated, group-specific transcription factor(s) recognizing arrays of inverted repeats of the element NGAAN. To date genes for two human factors, HSF1 and HSF2, have been isolated. To define their properties as well as the changes they undergo during heat stress activation, we prepared polyclonal antibodies to these factors. Using these tools, we have shown that human HeLa cells constitutively synthesize HSF1, but we were unable to detect HSF2. In unstressed cells HSF1 is present mainly in complexes with an apparent molecular mass of about 200 kDa, unable to bind to DNA. Heat treatment induces a shift in the apparent molecular mass of HSF1 to about 700 kDa, concomitant with the acquisition of DNA-binding ability. Cross-linking experiments suggest that this change in complex size may reflect the trimerization of monomeric HSF1. Human HSF1 expressed in Xenopus oocytes does not bind DNA, but derepression of DNA-binding activity, as well as oligomerization of HSF1, occurs during heat treatment at the same temperature at which hsp gene expression is induced in this organism, suggesting that a conserved Xenopus protein(s) plays a role in this regulation. Inactive HSF1 resides in the cytoplasm of human cells; on activation it rapidly translocates to a soluble nuclear fraction, and shortly thereafter it becomes associated with the nuclear pellet. On heat shock, activatable HSF1, which might already have been posttranslationally modified in the unstressed cell, undergoes further modification. These different process provide multiple points of regulation of hsp gene expression.

scholarly journals Characteristics of functional enrichment and gene expression level of human putative transcriptional target genes

2016 ◽  
Author(s):  
Naoki Osato
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Binding Sites ◽  
Target Genes ◽  
Functional Enrichment ◽  
Expression Level ◽  
Ctcf Binding ◽  
Cellular Functions ◽  
Median Expression ◽  
Transcriptional Target

AbstractBackgroundTranscriptional target genes show functional enrichment of genes. However, how many and how significantly transcriptional target genes include functional enrichments are still unclear. To address these issues, I predicted human transcriptional target genes using open chromatin regions, ChIP-seq data and DNA binding sequences of transcription factors in databases, and examined functional enrichment and gene expression level of putative transcriptional target genes.ResultsGene Ontology annotations showed four times larger numbers of functional enrichments in putative transcriptional target genes than gene expression information alone, independent of transcriptional target genes. To compare the number of functional enrichments of putative transcriptional target genes between cells or search conditions, I normalized the number of functional enrichment by calculating its ratios in the total number of transcriptional target genes. With this analysis, native putative transcriptional target genes showed the largest normalized number of functional enrichments, compared with target genes including 5 – 60% of randomly selected genes. The normalized number of functional enrichments was changed according to the criteria of enhancer-promoter interactions such as distance from transcriptional start sites and orientation of CTCF-binding sites. Forward-reverse orientation of CTCF-binding sites showed significantly higher normalized number of functional enrichments than the other orientations. Journal papers showed that the top five frequent functional enrichments were related to the cellular functions in the three cell types. The median expression level of transcriptional target genes changed according to the criteria of enhancer-promoter assignments (i.e. interactions) and was correlated with the changes of the normalized number of functional enrichments of transcriptional target genes.ConclusionsHuman putative transcriptional target genes showed significant functional enrichments. Functional enrichments were related to the cellular functions. The normalized number of functional enrichments of human putative transcriptional target genes changed according to the criteria of enhancer-promoter assignments and correlated with the median expression level of the target genes. These analyses and characters of human putative transcriptional target genes would be useful to examine the criteria of enhancer-promoter assignments and to predict the novel mechanisms and factors such as DNA binding proteins and DNA sequences of enhancer-promoter interactions.

Abstract 51: LincAQPEP, an Adipocyte-Specific Intergenic Noncoding RNA, Modulates Lipid Metabolism in Human Adipocytes

2015 ◽  
Vol 35 (suppl_1) ◽  
Author(s):  
Xuan Zhang ◽  
Chenyi Xue ◽  
Yumiao Han ◽  
Benjamin Garcia ◽  
Raymond Soccio ◽  
...  
Keyword(s):  
Gene Expression ◽  
Adipose Tissue ◽  
Transcription Start Site ◽  
Molecular Mechanisms ◽  
Ppar Gamma ◽  
Regulatory Function ◽  
Binding Motif ◽  
Start Site ◽  
Human Adipocytes ◽  
Transcription Start

Long intergenic noncoding RNAs (lincRNAs) have emerged as key mediators of cellular functions and are increasingly implicated in human diseases. Recently a set of lincRNAs was identified to regulate adipogenesis in mice and humans. Here we report that lincAQPEP, a lincRNA predominantly expressed in adipose tissue, is one of the most abundant lincRNAs detected in human fat by RNA sequencing. It is highly expressed in mature adipocytes but not detected in pre-adipocytes, monocytes or macrophages, indicating its adipocyte specificity. Interestingly, no syntenic or conserved RNA transcript was detected in mouse genome for lincAQPEP, suggesting it might be a human specific lincRNA. A binding motif for PPAR gamma (PPARγ), a critical transcription factor in adipogenesis, was found in the promoter region of lincAQPEP (-612 to -596 bp relative to transcription start site). In addition, chromatin immunoprecipitation sequencing confirmed high occupancy of PPARγ around lincAQPEP transcription start site in human adipose tissue and cultured adipocytes, suggesting PPARγ may mediate adipocyte-specific lincAQPEP transcription. LincAQPEP knockdown (~70%) by lentiviral-based short hairpin RNAs resulted in ~35% decrease in triglyceride content as well as 30-70% reduction of lipogenic gene expression (e.g. SREBP1, FASN, FABP4) in human adipocytes, suggesting a modulatory role of lincAQPEP in adipocyte function. To investigate the molecular mechanisms of its regulatory function, we performed RNA pulldown assays with biotinylated lincAQPEP and lincAQPEP antisense transcript (negative control), followed by mass spectrometry to discover potential lincAQPEP-interacting proteins. IGF2BP3 (Insulin-like growth factor 2 mRNA-binding protein 3), implicated in post-transcriptional regulation of gene expression, was identified as one of top candidates to physically bind lincAQPEP. Interestingly, lincAQPEP knockdown resulted in ~30% reduction in IGF2BP3 mRNA abundance. These data suggested that lincAQPEP may impact mRNA stability and translation of specific genes by interacting with IGF2BP3. In summary, our data indicate that lincAQPEP, an adipose-specific lincRNA, plays important roles in adipocyte biology likely through interaction with IGF2BP3.

Activation of human heat shock genes is accompanied by oligomerization, modification, and rapid translocation of heat shock transcription factor HSF1

1993 ◽  
Vol 13 (4) ◽  
pp. 2486-2496
Author(s):  
R Baler ◽  
G Dahl ◽  
R Voellmy
Keyword(s):  
Gene Expression ◽  
Heat Treatment ◽  
Transcription Factor ◽  
Heat Shock ◽  
Dna Binding ◽  
Molecular Mass ◽  
Polyclonal Antibodies ◽  
Protein S ◽  
Apparent Molecular Mass ◽  
Nuclear Fraction

Transcriptional activity of heat shock (hsp) genes is controlled by a heat-activated, group-specific transcription factor(s) recognizing arrays of inverted repeats of the element NGAAN. To date genes for two human factors, HSF1 and HSF2, have been isolated. To define their properties as well as the changes they undergo during heat stress activation, we prepared polyclonal antibodies to these factors. Using these tools, we have shown that human HeLa cells constitutively synthesize HSF1, but we were unable to detect HSF2. In unstressed cells HSF1 is present mainly in complexes with an apparent molecular mass of about 200 kDa, unable to bind to DNA. Heat treatment induces a shift in the apparent molecular mass of HSF1 to about 700 kDa, concomitant with the acquisition of DNA-binding ability. Cross-linking experiments suggest that this change in complex size may reflect the trimerization of monomeric HSF1. Human HSF1 expressed in Xenopus oocytes does not bind DNA, but derepression of DNA-binding activity, as well as oligomerization of HSF1, occurs during heat treatment at the same temperature at which hsp gene expression is induced in this organism, suggesting that a conserved Xenopus protein(s) plays a role in this regulation. Inactive HSF1 resides in the cytoplasm of human cells; on activation it rapidly translocates to a soluble nuclear fraction, and shortly thereafter it becomes associated with the nuclear pellet. On heat shock, activatable HSF1, which might already have been posttranslationally modified in the unstressed cell, undergoes further modification. These different process provide multiple points of regulation of hsp gene expression.

scholarly journals Electroacupuncture Mimics Exercise-Induced Changes in Skeletal Muscle Gene Expression in Women With Polycystic Ovary Syndrome

2020 ◽  
Vol 105 (6) ◽  
pp. 2027-2041 ◽  
Author(s):  
Anna Benrick ◽  
Nicolas J Pillon ◽  
Emma Nilsson ◽  
Eva Lindgren ◽  
Anna Krook ◽  
...  
Keyword(s):  
Gene Expression ◽  
Skeletal Muscle ◽  
Polycystic Ovary Syndrome ◽  
Glucose Uptake ◽  
Molecular Mechanisms ◽  
Polycystic Ovary ◽  
Glycogen Synthesis ◽  
Whole Body ◽  
Ovary Syndrome ◽  
Altered Expression

Abstract Context Autonomic nervous system activation mediates the increase in whole-body glucose uptake in response to electroacupuncture but the mechanisms are largely unknown. Objective To identify the molecular mechanisms underlying electroacupuncture-induced glucose uptake in skeletal muscle in insulin-resistant overweight/obese women with and without polycystic ovary syndrome (PCOS). Design/Participants In a case-control study, skeletal muscle biopsies were collected from 15 women with PCOS and 14 controls before and after electroacupuncture. Gene expression and methylation was analyzed using Illumina BeadChips arrays. Results A single bout of electroacupuncture restores metabolic and transcriptional alterations and induces epigenetic changes in skeletal muscle. Transcriptomic analysis revealed 180 unique genes (q &lt; 0.05) whose expression was changed by electroacupuncture, with 95% of the changes towards a healthier phenotype. We identified DNA methylation changes at 304 unique sites (q &lt; 0.20), and these changes correlated with altered expression of 101 genes (P &lt; 0.05). Among the 50 most upregulated genes in response to electroacupuncture, 38% were also upregulated in response to exercise. We identified a subset of genes that were selectively altered by electroacupuncture in women with PCOS. For example, MSX1 and SRNX1 were decreased in muscle tissue of women with PCOS and were increased by electroacupuncture and exercise. siRNA-mediated silencing of these 2 genes in cultured myotubes decreased glycogen synthesis, supporting a role for these genes in glucose homeostasis. Conclusion Our findings provide evidence that electroacupuncture normalizes gene expression in skeletal muscle in a manner similar to acute exercise. Electroacupuncture might therefore be a useful way of assisting those who have difficulties performing exercise.

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