Chromatin immunoprecipitation of transcription factors and histone modifications in Comma-Dβ mammary epithelial cells.
Ontology highlight
ABSTRACT:
SUBMITTER: Holliday H
PROVIDER: S-EPMC8113982 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Histone post-translational modifications (PTMs), such as acetylation, methylation and phosphorylation, are dynamically regulated by a series of enzymes that add or remove these marks in response to signals received by the cell. These PTMS are key contributors to the regulation of processes such as gene expression control and DNA repair. Chromatin immunoprecipitation (chIP) has been an instrumental approach for dissecting the abundance and localization of many histone PTMs throughout the genome in response to diverse perturbations to the cell. Here, a versatile method for performing chIP of post-translationally modified histones from the budding yeast Saccharomyces cerevisiae (S. cerevisiae) is described. This method relies on crosslinking of proteins and DNA using formaldehyde treatment of yeast cultures, generation of yeast lysates by bead beating, solubilization of chromatin fragments by micrococcal nuclease, and immunoprecipitation of histone-DNA complexes. DNA associated with the histone mark of interest is purified and subjected to quantitative PCR analysis to evaluate its enrichment at multiple loci throughout the genome. Representative experiments probing the localization of the histone marks H3K4me2 and H4K16ac in wildtype and mutant yeast are discussed to demonstrate data analysis and interpretation. This method is suitable for a variety of histone PTMs and can be performed with different mutant strains or in the presence of diverse environmental stresses, making it an excellent tool for investigating changes in chromatin dynamics under different conditions.
| S-EPMC5908393 | biostudies-literature
Project description:Previous studies have demonstrated that miR-34a is a direct transcriptional target of tumor suppressor p53 and plays a crucial role in p53-mediated biological processes, such as cell cycle arrest, apoptosis and senescence. However, the role of p53 phosphorylation at Ser15 and histone modifications in ionizing radiation (IR)-induced miR-34a transcription in human mammary epithelial cells remains unknown. The present study showed that IR triggers miR-34a induction in rat mammary gland tissue and human mammary epithelial cells in a dose- and time-dependent fashion. Gene copy number and CpG methylation exhibit no effect on IR-inducible miR-34a expression, while the levels of phosphorylated p53 at Ser15 are markedly elevated in human mammary epithelial cells 96 h post-IR, which correlates with IR-inducible miR-34a transcription and the p38 MAPK pathway. Conversely, suppression of p38 MAPK with SB239063 inhibits IR-induced p53 phosphorylation at Ser15 and miR-34a expression in a dose-dependent manner. Our study found that wild-type p53 is enriched at miR-34a promoter, and luciferase activity of miR-34a promoter reporter is attenuated by either mutant p53 (Ser15Ala) or mutant miR-34a promoter. Furthermore, IR also triggers phosphorylation, tri-methylation and acetylation of histone H3 and acetylation of histone H4, which correlates with IR-inducible miR-34a transcription, while SAHA potentiates IR-inducible miR-34a expression. Moreover, acetyl-histone H3 is significantly enriched at miR-34a promoter in IR-exposed HMEC cells. Yet, we show that there is no correlation between IR-inducible miR-34a expression and IR-induced rapid and transient G 2/M arrest. In sum, our novel data for the first time demonstrate that IR-induced p53 Ser15 phosphorylation via p38 MAPK is essential for its functional regulation of IR-inducible miR-34a transcription in human mammary epithelial cells, and that histone modifications may also play a key role in IR-inducible miR-34a expression.
| S-EPMC3737310 | biostudies-literature
Project description:Chromatin immunoprecipitation coupled to next-generation sequencing (ChIP-seq) has served as the central method for the study of histone modifications for the past decade. In ChIP-seq analyses, antibodies selectively capture nucleosomes bearing a modification of interest and the associated DNA is then mapped to the genome to determine the distribution of the mark. This approach has several important drawbacks: (i) ChIP interpretation necessitates the assumption of perfect antibody specificity, despite growing evidence that this is often not the case. (ii) Common methods for evaluating antibody specificity in other formats have little or no bearing on specificity within a ChIP experiment. (iii) Uncalibrated ChIP is reported as relative enrichment, which is biologically meaningless outside the experimental reference frame defined by a discrete immunoprecipitation (IP), thus preventing facile comparison across experimental conditions or modifications. (iv) Differential library amplification and loading onto next-generation sequencers, as well as computational normalization, can further compromise quantitative relationships that may exist between samples. Consequently, the researcher is presented with a series of potential pitfalls and is blind to nearly all of them. Here we provide a detailed protocol for internally calibrated ChIP (ICeChIP), a method we recently developed to resolve these problems by spike-in of defined nucleosomal standards within a ChIP procedure. This protocol is optimized for specificity and quantitative power, allowing for measurement of antibody specificity and absolute measurement of histone modification density (HMD) at genomic loci on a biologically meaningful scale enabling unambiguous comparisons. We provide guidance on optimal conditions for next-generation sequencing (NGS) and instructions for data analysis. This protocol takes between 17 and 18 h, excluding time for sequencing or bioinformatic analysis. The ICeChIP procedure enables accurate measurement of histone post-translational modifications (PTMs) genome-wide in mammalian cells as well as Drosophila melanogaster and Caenorhabditis elegans, indicating suitability for use in eukaryotic cells more broadly.
| S-EPMC7158589 | biostudies-literature
Project description:Differentiated HC-11 cells ectopically expressing progesterone receptor (PR) were used to explore the molecular mechanisms by which progesterone suppresses ?-casein gene transcription induced by prolactin (PRL) and glucocorticoids in the mammary gland. As detected by chromatin immunoprecipitation assays, treatment of cells with the progestin agonist R5020 induced a rapid recruitment (5 min) of PR to the proximal promoter (-235 bp) and distal enhancer (-6 kb upstream of transcription start site) of ?-casein. PR remained bound for 4 h and was dissociated by 24 h after treatment. Despite efficient binding, the hormone agonist-occupied PR did not stimulate transcription of the ?-casein gene. Recruitment of signal transducer and activator of transcription 5a, glucocorticoid receptor, and the CCAAT enhancer binding protein ? to the enhancer and proximal promoter of ?-casein induced by PRL and glucocorticoids was blocked by progestin cotreatment, whereas PR binding was induced under these conditions. PRL/glucocorticoid-induced histone acetylation and the recruitment of the coactivator p300 and RNA polymerase II required for gene activation were also inhibited by progestin. In addition, progestin prevented dissociation of the corepressors Yin and Yang 1 and histone deacetylase 3 from the promoter, and demethylation of lysine 9 of histone 3 induced by PRL and glucocorticoids. These studies are consistent with the conclusion that progesterone interferes with PRL/glucocorticoid induction of ?-casein transcription by a physical interaction of PR with the promoter and enhancer that blocks assembly of a transcriptional activation complex and dissociation of corepressors and promotes repressive chromatin modifications. These studies define a novel mechanism of steroid receptor-mediated transcriptional repression of a physiologically important gene in mammary gland development and differentiation.
| S-EPMC3386529 | biostudies-literature
Project description:In this report we describe a chromatin immunoprecipitation (ChIP) protocol for two fully sequenced model diatom species Phaeodactylum tricornutum and Thalassiosira pseudonana. This protocol allows the extraction of satisfactory amounts of chromatin and gives reproducible results. We coupled the ChIP assay with real time quantitative PCR. Our results reveal that the two major histone marks H3K4me2 and H3K9me2 exist in P. tricornutum and T. pseudonana. As in other eukaryotes, H3K4me2 marks active genes whereas H3K9me2 marks transcriptionally inactive transposable elements. Unexpectedly however, T. pseudonana housekeeping genes also show a relative enrichment of H3K9me2. We also discuss optimization of the procedure, including growth conditions, cross linking and sonication. Validation of the protocol provides a set of genes and transposable elements that can be used as controls for studies using ChIP in each diatom species. This protocol can be easily adapted to other diatoms and eukaryotic phytoplankton species for genetic and biochemical studies.
| S-EPMC3546051 | biostudies-literature
Project description:Interactions between DNA and proteins located in the cell nucleus play an important role in controlling physiological processes by specifying, augmenting and regulating context-specific transcription events. Chromatin immunoprecipitation (ChIP) is a widely used methodology to study DNA-protein interactions and has been successfully used in various cell types for over three decades. More recently, by combining ChIP with genomic screening technologies and Next Generation Sequencing (e.g. ChIP-seq), it has become possible to profile DNA-protein interactions (including covalent histone modifications) across entire genomes. However, the applicability of ChIP-chip and ChIP-seq has rarely been extended to non-model species because of a number of technical challenges. Here we report a method that can be used to identify genome wide covalent histone modifications in a group of non-model fruit fly species (Diptera: Tephritidae). The method was developed by testing and refining protocols that have been used in model organisms, including Drosophila melanogaster. We demonstrate that this method is suitable for a group of economically important pest fruit fly species, viz., Bactrocera dorsalis, Ceratitis capitata, Zeugodacus cucurbitae and Bactrocera tryoni. We also report an example ChIP-seq dataset for B. tryoni, providing evidence for histone modifications in the genome of a tephritid fruit fly for the first time. Since tephritids are major agricultural pests globally, this methodology will be a valuable resource to study taxa-specific evolutionary questions and to assist with pest management. It also provides a basis for researchers working with other non-model species to undertake genome wide DNA-protein interaction studies.
| S-EPMC5854383 | biostudies-literature
Project description:According to the transcription factory model, localized transcription sites composed of immobilized polymerase molecules transcribe chromatin by reeling it through the transcription site and extruding it to form a surrounding domain of recently transcribed decondensed chromatin. Although transcription sites have been identified in various cells, surrounding domains of recently transcribed decondensed chromatin have not. We report evidence that transcription sites associated with a tandem gene array in mouse cells are indeed surrounded by or adjacent to a domain of decondensed chromatin composed of sequences from the gene array. Formation of this decondensed domain requires transcription and topoisomerase IIalpha activity. The decondensed domain is enriched for the trimethyl H3K36 mark that is associated with recently transcribed chromatin in yeast and several mammalian systems. Consistent with this, chromatin immunoprecipitation demonstrates a comparable enrichment of this mark in transcribed sequences at the tandem gene array. These results provide new support for the pol II factory model, in which an immobilized polymerase molecule extrudes decondensed, transcribed sequences into its surroundings.
| S-EPMC2064357 | biostudies-literature
Project description:Smads are intracellular transducers for TGF-beta superfamily ligands, but little is known about the mechanism by which complexes of receptor-phosphorylated Smad2 and Smad4 regulate transcription. Using an in vitro transcription system, we have discovered that, unlike most transcription factors that are sufficient to recruit the basal transcription machinery and therefore activate transcription on both naked DNA and chromatin templates, the Smads only activate transcription from chromatin templates. We demonstrate that Smad2-mediated transcription requires the histone acetyltransferase, p300. Smad2-recruited p300 exhibits an altered substrate specificity, specifically acetylating nucleosomal histone H3 at lysines 9 and 18, and these modifications are also detected on an endogenous Smad2-dependent promoter in a ligand-induced manner. Furthermore, we show that endogenous Smad2 interacts with the SWI/SNF ATPase, Brg1, in a TGF-beta-dependent manner, and demonstrate that Brg1 is recruited to Smad2-dependent promoters and is specifically required for TGF-beta-induced expression of endogenous Smad2 target genes. Our data indicate that the Smads define a new class of transcription factors that absolutely require chromatin to assemble the basal transcription machinery and activate transcription.
| S-EPMC1589990 | biostudies-literature
Project description:Genome-wide location analysis (ChIP-chip, ChIP-PET) is a powerful technique to study mammalian transcriptional regulation. In order to obtain a basic understanding of the location data generated for mammalian transcription factors and potential issues in their analysis, we conducted a comparative study of eight independent ChIP experiments involving six different transcription factors in human and mouse. Our cross-study comparisons, to the best of our knowledge the first to analyze multiple datasets, revealed the importance of carefully chosen genomic controls in the de novo identification of key transcription factor binding motifs, raised issues about the interpretation of ubiquitously occurring sequence motifs, and demonstrated the clustering tendency of protein-binding regions for certain transcription factors.
| S-EPMC1669715 | biostudies-literature
Project description:The goal of this study was to determine the genome wide binding sites of the E-protein transcription factor HEB in mammary epithelial cells, and to delineate the chromatin context of these regions. ChIP-seq for HEB, H3K4Me3, H3K27Ac, and H3K27Me was performed in Comma-Dβ cells in quadruplicate.
2020-05-07 | GSE149967 | GEO