221 EPIGENETIC DYNAMICS OF PLURIPOTENCY GENES IN THE CONTEXT OF DIFFERENTIATION AND NUCLEAR REPROGRAMMING DETERMINED BY Q2ChIP, A QUICK AND QUANTITATIVE CHROMATIN IMMUNOPRECIPITATION TECHNIQUE APPLICABLE TO SMALL CELL SAMPLES

2007 ◽  
Vol 19 (1) ◽  
pp. 227 ◽  
Author(s):  
J. A. Dahl ◽  
C. K. Taranger ◽  
P. Collas
Keyword(s):  
Gene Expression ◽  
Chromatin Immunoprecipitation ◽  
Regulation Of Gene Expression ◽  
Cell Extract ◽  
Proximal Promoter ◽  
Pcr Analysis ◽  
Cross Linking ◽  
Nuclear Reprogramming ◽  
Distal Enhancer ◽  
Developmentally Regulated

Interactions between proteins and DNA are essential for cellular functions such as genomic stability, DNA replication and repair, chromosome segregation, transcription, and epigenetic silencing of gene expression. Chromatin immunoprecipitation (ChIP) is a key technique for mapping histone modifications and transcription factor binding on DNA and thereby unraveling the role of epigenetics in the regulation of gene expression. Current ChIP protocols require extensive sample handling and large numbers of cells (5-10 million). primarily owing to ample loss of material during the procedure. We altered critical steps of conventional ChIP to develop a quick and quantitative (Q2) ChIP assay suitable for cell numbers 100- to 1000-fold lower than those required for conventional ChIP. Key modifications of the ChIP procedure include (i) formaldehyde DNA–protein cross-linking in suspended cells, (ii) cross-linking in the presence of 20 mM sodium butyrate to enhance specificity of precipitation of acetylated histones, (iii) transfer of washed precipitated immune complexes to a clean tube ('tube shift') to increase ChIP specificity by virtually eliminating nonspecifically bound chromatin, and (iv) combination of cross-link reversal, protein digestion, and DNA elution into a single 2-h step. We used Q2ChIP to monitor changes in 6 histone H3 modifications on the human developmentally regulated genes OCT4 (POU5F1), NANOG, and LMNA (lamin A) in the context of retinoic acid (RA)-mediated differentiation of embryonal carcinoma cells and upon reprogramming of kidney epithelial 293T cells to pluripotency in carcinoma cell extract (Taranger et al. 2005 Mol. Biol. Cell 16, 5719–5735). Real-time PCR analysis of precipitated DNA unravels an unexpected two-step heterochromatin assembly elicited by RA on the OCT4 proximal promoter, proximal enhancer, and distal enhancer, and on the NANOG promoter, whereby methylation of H3K9 and H3K27 is followed by H3K9 deacetylation. H3K4 di- and trimethylation remain relatively unaffected by RA treatment. In contrast, reprogramming of 293T cells in carcinoma extract promotes assembly of histone marks characteristic of transcriptional induction of OCT4 and NANOG, such as acetylation and demethylation of H3K9. The results argue toward ordered chromatin repackaging at developmentally regulated promoters upon differentiation or, conversely, nuclear reprogramming to pluripotency.

scholarly journals Epigenetic Changes of the Cyp11a1 Promoter Region in Granulosa Cells Undergoing Luteinization During Ovulation in Female Rats

Endocrinology ◽  
2016 ◽  
Vol 157 (9) ◽  
pp. 3344-3354 ◽  
Author(s):  
Maki Okada ◽  
Lifa Lee ◽  
Ryo Maekawa ◽  
Shun Sato ◽  
Takuya Kajimura ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Granulosa Cells ◽  
Chromatin Immunoprecipitation ◽  
Promoter Region ◽  
Binding Protein ◽  
Female Rats ◽  
Proximal Promoter ◽  
Lh Surge ◽  
Enhancer Binding Protein

The ovulatory LH surge induces rapid up-regulation of Cyp11a1 in granulosa cells (GCs) undergoing luteinization during ovulation. This study investigated in vivo whether epigenetic controls including histone modifications and DNA methylation in the promoter region are associated with the rapid increase of Cyp11a1 gene expression after LH surge. GCs were obtained from rats treated with equine chorionic gonadotropin (CG) before (0 h) and 4 h and 12 h after human (h)CG injection. Cyp11a1 mRNA levels rapidly increased after hCG injection, reached a peak at 4 hours, and then remained elevated until 12 hours. DNA methylation status in the Cyp11a1 proximal promoter region was hypomethylated and did not change at any of the observed times after hCG injection. Chromatin immunoprecipitation assays revealed that the levels of trimethylation of lysine 4 on histone H3 (H3K4me3), an active mark for transcription, increased, whereas the levels of H3K9me3 and H3K27me3, which are marks associated with repression of transcription, decreased in the Cyp11a1 proximal promoter after hCG injection. Chromatin condensation, which was analyzed using deoxyribonuclease I, decreased in the Cyp11a1 proximal promoter after hCG injection. Chromatin immunoprecipitation assays also showed that the binding activity of CAATT/enhancer-binding protein-β to the Cyp11a1 proximal promoter increased after hCG injection. Luciferase assays revealed that the CAATT/enhancer-binding protein-β-binding site had transcriptional activity and contributed to basal and cAMP-induced Cyp11a1 expression. These results suggest that changes in histone modification and chromatin structure in the Cyp11a1 proximal promoter are involved in the rapid increase of Cyp11a1 gene expression in GCs undergoing luteinization during ovulation.

scholarly journals Expresso: A database and web server for exploring the interaction of transcription factors and their target genes in Arabidopsis thaliana using ChIP-Seq peak data

F1000Research ◽  
2017 ◽  
Vol 6 ◽  
pp. 372 ◽  
Author(s):  
Delasa Aghamirzaie ◽  
Karthik Raja Velmurugan ◽  
Shuchi Wu ◽  
Doaa Altarawy ◽  
Lenwood S. Heath ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Transcription Factors ◽  
Chromatin Immunoprecipitation ◽  
Target Genes ◽  
Regulation Of Gene Expression ◽  
Data Sets ◽  
Motif Analysis ◽  
Chromatin Immunoprecipitation Sequencing

Motivation: The increasing availability of chromatin immunoprecipitation sequencing (ChIP-Seq) data enables us to learn more about the action of transcription factors in the regulation of gene expression. Even though in vivo transcriptional regulation often involves the concerted action of more than one transcription factor, the format of each individual ChIP-Seq dataset usually represents the action of a single transcription factor. Therefore, a relational database in which available ChIP-Seq datasets are curated is essential. Results: We present Expresso (database and webserver) as a tool for the collection and integration of available Arabidopsis ChIP-Seq peak data, which in turn can be linked to a user’s gene expression data. Known target genes of transcription factors were identified by motif analysis of publicly available GEO ChIP-Seq data sets. Expresso currently provides three services: 1) Identification of target genes of a given transcription factor; 2) Identification of transcription factors that regulate a gene of interest; 3) Computation of correlation between the gene expression of transcription factors and their target genes. Availability: Expresso is freely available at http://bioinformatics.cs.vt.edu/expresso/

scholarly journals Epigenetic Reprogramming of OCT4 and NANOG Regulatory Regions by Embryonal Carcinoma Cell Extract

2007 ◽  
Vol 18 (5) ◽  
pp. 1543-1553 ◽  
Author(s):  
Christel T. Freberg ◽  
John Arne Dahl ◽  
Sanna Timoskainen ◽  
Philippe Collas
Keyword(s):  
Transcriptional Activation ◽  
Transcriptional Repression ◽  
Embryonal Carcinoma ◽  
Cell Extract ◽  
Epigenetic Reprogramming ◽  
Proximal Promoter ◽  
Embryonal Carcinoma Cell ◽  
Distal Enhancer ◽  
Regulatory Regions ◽  
Molecular Events

Analyses of molecular events associated with reprogramming somatic nuclei to pluripotency are scarce. We previously reported the reprogramming of epithelial cells by extract of undifferentiated embryonal carcinoma (EC) cells. We now demonstrate reprogramming of DNA methylation and histone modifications on regulatory regions of the developmentally regulated OCT4 and NANOG genes by exposure of 293T cells to EC cell extract. OCT4 and NANOG are transcriptionally up-regulated and undergo mosaic cytosine-phosphate-guanosine demethylation. OCT4 demethylation occurs as early as week 1, is enhanced by week 2, and is most prominent in the proximal promoter and distal enhancer. Targeted OCT4 and NANOG demethylation does not occur in 293T extract-treated cells. Retinoic acid-mediated differentiation of reprogrammed cells elicits OCT4 promoter remethylation and transcriptional repression. Chromatin immunoprecipitation analyses of lysines K4, K9, and K27 of histone H3 on OCT4 and NANOG indicate that primary chromatin remodeling determinants are acetylation of H3K9 and demethylation of dimethylated H3K9. H3K4 remains di- and trimethylated. Demethylation of trimethylated H3K9 and H3K27 also occurs; however, trimethylation seems more stable than dimethylation. We conclude that a central epigenetic reprogramming event is relaxation of chromatin at loci associated with pluripotency to create a conformation compatible with transcriptional activation.

scholarly journals A ChIP on the shoulder? Chromatin immunoprecipitation and validation strategies for ChIP antibodies

F1000Research ◽  
2015 ◽  
Vol 4 ◽  
pp. 235 ◽  
Author(s):  
Fiona C. Wardle ◽  
Haihan Tan
Keyword(s):  
Gene Expression ◽  
Transcriptional Regulation ◽  
Chromatin Immunoprecipitation ◽  
Regulation Of Gene Expression ◽  
Antigen Recognition ◽  
Validation Methods

Chromatin immunoprecipitation (ChIP) is a technique widely used in the study of epigenetics and transcriptional regulation of gene expression. However, its antibody-centric nature exposes it to similar challenges faced by other antibody-based procedures, of which the most prominent are issues of specificity and affinity in antigen recognition. As with other techniques that make use of antibodies, recent studies have shown the need for validation of ChIP antibodies in order to be sure they recognize the advertised protein or epitope. We summarize here the issues surrounding ChIP antibody usage, and highlight the toolkit of validation methods that can be employed by investigators looking to appraise these reagents.

Exploiting XGBoost For Predicting Enhancer-Promoter Interactions.

2020 ◽  
Vol 15 ◽  
Author(s):  
Xiaojuan Yu ◽  
Jianguo Zhou ◽  
Mingming Zhao ◽  
Chao Yi ◽  
Qing Duan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Genetic Basis ◽  
High Throughput Sequencing ◽  
The Other ◽  
Proximal Promoter ◽  
Sequence Feature ◽  
Distal Enhancer ◽  
Recent Emergence ◽  
Insight Into

: It is well-known that gene expression and disease control are co-regulated by the interaction between the distal enhancer and the proximal promoter, and the study of enhancer promoter interactions (EPIs) can help us to gain insight into the genetic basis of diseases. Although the recent emergence of some high-throughput sequencing methods have given us a deeper understanding of EPIs, accurate prediction of EPIs still have some limitations. In this paper, we trained a XGBoost based model and introduced two sets of features (i.e. epigenomic and sequence feature) to predict the interactions between the enhancer and the promoter in different cell lines. We compared XGBoost with the other four methods. Extensive experimental results have shown that XGBoost based method is effective in predicting EPIs across three cell lines. Especially epigenomic and sequence features can boost prediction.

scholarly journals Characterization of Soluble Sugar Content, Related Enzyme Activity and Gene Expression in the Fruits of ‘Minihyang’ Mandarin on Different Rootstocks

Horticulturae ◽  
2022 ◽  
Vol 8 (1) ◽  
pp. 47
Author(s):  
Ha Rim Hong ◽  
Eun Ui Oh ◽  
Seung Gab Han ◽  
Su Hyun Yun ◽  
Ho Bang Kim ◽  
...  
Keyword(s):  
Gene Expression ◽  
Enzyme Activity ◽  
Sugar Content ◽  
Soluble Sugar ◽  
Regulation Of Gene Expression ◽  
Fruit Size ◽  
Pcr Analysis ◽  
Total Sugar Content ◽  
Significant Difference ◽  
Days After Anthesis

‘Minihyang’ mandarin bears fruits with small size and high sugar content. ‘Minihyang’ mandarin grafted on trifoliate orange (TO) tends to be vigorous and develops water sprout open. It is associated with insufficient floral differentiation and fruit set. Recently, the use of Flying Dragon (FD) as rootstock with a high dwarf effect has been proposed to improve this situation. Therefore, this study was conducted to evaluate the effect of two different rootstock genotypes on tree growth, fruit yield, and fruit quality at the physiological, biochemical, and molecular levels. As a result of the study, in FD, tree vigor was stably maintained, fruit size was large, and the sugar content was high compared to the TO. Fructose, glucose, and sucrose of fruit continued to increase from development to maturity. In particular, fructose and sucrose were significantly higher in the fruits of the FD than those in TO at 150 and 220 days after anthesis. The total sugar content was also significantly higher in the fruit of the FD. The activities of SPS and SS associated with sucrose synthesis tended to be increased during the fruit maturity season, but there was no significant difference between the two rootstocks. On the other hand, the activities of SS and AI breaking down sucrose were high in FD at 150 and 220 days after anthesis. These results suggest that the unloading of sucrose might be increased and affect the sugar content. However, the results of real-time PCR analysis of gene expression related to sucrose metabolism did not show an association with changes in enzyme activity affecting sugar content. Therefore, further detailed studies on the process after the regulation of gene expression are likely to be needed.

scholarly journals The chromatin landscape of the casein gene locus

2012 ◽  
Vol 10 (1) ◽  
Author(s):  
Monique Rijnkels ◽  
Elena Kabotyanski ◽  
Amy Shore ◽  
Jeffrey M. Rosen
Keyword(s):  
Gene Expression ◽  
Hormonal Regulation ◽  
Dna Looping ◽  
Proximal Promoter ◽  
Specific Factor ◽  
Distal Enhancer ◽  
Specific Expression ◽  
Casein Gene ◽  
Cooperative Effects ◽  
Conserved Sequence

AbstractFor several decades, the regulation of casein gene expression by the lactogenic hormones, prolactin and glucocorticoids, has provided an excellent model system in which to study how steroid and peptide hormones regulate gene expression. Early studies of casein gene regulation defined conserved sequence elements in the 5′ flanking region of these genes, including one of which was identified as a γ-interferon activation sequence (GAS). Although this site was thought to interact with a mammary gland-specific factor, purification and cloning of this factor by Bernd Groner and his colleagues revealed it was instead a new member of the signal transducers and activators of transcription family, Stat5, which was expressed in many tissues. The exquisite tissue-specific expression of the casein genes was subsequently shown to depend not on a single transcription factor but on composite response elements that interacted with a number of ubiquitous transcription factors in response to the combinatorial effects of peptide and steroid hormone signaling. More recent studies have defined cooperative effects of prolactin and glucocorticoids as well as antagonistic effects of progesterone on the chromatin structure of both the casein gene proximal promoter region as well as a distal enhancer. Local chromatin modifications as well as long-range interactions facilitated by DNA looping are required for the hormonal regulation of β-

MafA, NeuroD1, and HNF1β synergistically activate Slc2a2 (Glut2) gene in β-cells

2021 ◽  
Author(s):  
Yuka Ono ◽  
Kohsuke Kataoka
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Transcriptional Activation ◽  
Glucose Transporter ◽  
Proximal Promoter ◽  
Luciferase Reporter ◽  
Distal Enhancer ◽  
Β Cells ◽  
Β Cell ◽  
Target Sites

Glucose transporter type 2 (GLUT2), encoded by the SLC2A2 gene, is an essential component of glucose-stimulated insulin secretion in pancreatic islet β-cells. Like that of the gene encoding insulin, expression of the SLC2A2 gene expression is closely linked to β-cell functionality in rodents, but the mechanism by which β-cell-specific expression of SLC2A2 is controlled remains unclear. In this report, to identify putative enhancer elements of the mouse Slc2a2 gene, we examined evolutional conservation of the nucleotide sequence of its genomic locus, together with ChIP-seq data of histone modifications and various transcription factors published in previous studies. Using luciferase reporter assays, we found that an evolutionarily conserved region located approximately 40 kbp downstream of the transcription start site of Slc2a2 functions as an active enhancer in the MIN6 β-cell line. We also found that three β-cell-enriched transcription factors, MafA, NeuroD1, and HNF1β, synergistically activate transcription through this 3’ downstream distal enhancer (ECR3’) and the proximal promoter region of the gene. Our data also indicate that the simultaneous binding of HNF1β to its target sites within the promoter and ECR3’ of Slc2a2 is indispensable for transcriptional activation, and that binding of MafA and NeuroD1 to their respective target sites within the ECR3’ enhances transcription. Co-immunoprecipitation experiments suggested that MafA, NeuroD1, and HNF1β interact with each other. Overall, these results suggest that promoter-enhancer communication through MafA, NeuroD1, and HNF1β is critical for Slc2a2 gene expression. These findings provide clues to help elucidate the mechanism of regulation of Slc2a2 gene expression in β-cells.

scholarly journals Analysis of gene–environment interactions in postnatal development of the mammalian intestine

2015 ◽  
Vol 112 (7) ◽  
pp. 1929-1936 ◽  
Author(s):  
Seth Rakoff-Nahoum ◽  
Yong Kong ◽  
Steven H. Kleinstein ◽  
Sathish Subramanian ◽  
Philip P. Ahern ◽  
...  
Keyword(s):  
Gene Expression ◽  
Postnatal Development ◽  
Muscle Development ◽  
Regulation Of Gene Expression ◽  
Microbial Colonization ◽  
Double Knockout ◽  
Developmentally Regulated ◽  
Gene Environment ◽  
Intestinal Physiology ◽  
Myeloid Differentiation Factor

Unlike mammalian embryogenesis, which takes place in the relatively predictable and stable environment of the uterus, postnatal development can be affected by a multitude of highly variable environmental factors, including diet, exposure to noxious substances, and microorganisms. Microbial colonization of the intestine is thought to play a particularly important role in postnatal development of the gastrointestinal, metabolic, and immune systems. Major changes in environmental exposure occur right after birth, upon weaning, and during pubertal maturation into adulthood. These transitions include dramatic changes in intestinal contents and require appropriate adaptations to meet changes in functional demands. Here, we attempt to both characterize and provide mechanistic insights into postnatal intestinal ontogeny. We investigated changes in global intestinal gene expression through postnatal developmental transitions. We report profound alterations in small and large intestinal transcriptional programs that accompany both weaning and puberty in WT mice. Using myeloid differentiation factor 88 (MyD88)/TIR-domain-containing adapter-inducing interferon-β (TRIF) double knockout littermates, we define the role of toll-like receptors (TLRs) and interleukin (IL)-1 receptor family member signaling in postnatal gene expression programs and select ontogeny-specific phenotypes, such as vascular and smooth muscle development and neonatal epithelial and mast cell homeostasis. Metaanalysis of the effect of the microbiota on intestinal gene expression allowed for mechanistic classification of developmentally regulated genes by TLR/IL-1R (TIR) signaling and/or indigenous microbes. We find that practically every aspect of intestinal physiology is affected by postnatal transitions. Developmental timing, microbial colonization, and TIR signaling seem to play distinct and specific roles in regulation of gene-expression programs throughout postnatal development.

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