Dual Cross-Linking Chromatin Immunoprecipitation Protocol for Next-Generation Sequencing (ChIPseq) in Macrophages

2019 ◽  
pp. 87-98
Author(s):  
David A. Rollins ◽  
Inez Rogatsky
Keyword(s):  
Next Generation Sequencing ◽  
Chromatin Immunoprecipitation ◽  
Cross Linking ◽  
Next Generation ◽  
Generation Sequencing

scholarly journals Genome-Wide Chromatin Immunoprecipitation Sequencing Analysis of the Penicillium chrysogenum Velvet Protein PcVelA Identifies Methyltransferase PcLlmA as a Novel Downstream Regulator of Fungal Development

mSphere ◽  
2016 ◽  
Vol 1 (4) ◽  
Author(s):  
Kordula Becker ◽  
Sandra Ziemons ◽  
Katharina Lentz ◽  
Michael Freitag ◽  
Ulrich Kück
Keyword(s):  
Next Generation Sequencing ◽  
Secondary Metabolism ◽  
Filamentous Fungi ◽  
Chromatin Immunoprecipitation ◽  
Next Generation ◽  
Content Type ◽  
Major Interest ◽  
Lactam Antibiotic ◽  
Velvet Complex ◽  
Generation Sequencing

ABSTRACT Filamentous fungi are of major interest for biotechnological and pharmaceutical applications. This is due mainly to their ability to produce a wide variety of secondary metabolites, many of which are relevant as antibiotics. One of the most prominent examples is penicillin, a β-lactam antibiotic that is produced on the industrial scale by fermentation of P. chrysogenum. In recent years, the multisubunit protein complex velvet has been identified as one of the key regulators of fungal secondary metabolism and development. However, until recently, only a little has been known about how velvet mediates regulation at the molecular level. To address this issue, we performed ChIP-seq (chromatin immunoprecipitation in combination with next-generation sequencing) on and follow-up analysis of PcVelA, the core component of the velvet complex in P. chrysogenum. We demonstrate direct involvement of velvet in transcriptional control and present the putative methyltransferase PcLlmA as a new downstream factor and interaction partner of PcVelA. Penicillium chrysogenum is the sole industrial producer of the β-lactam antibiotic penicillin, which is the most commonly used drug for treating bacterial infections. In P. chrysogenum and other filamentous fungi, secondary metabolism and morphogenesis are controlled by the highly conserved multisubunit velvet complex. Here we present the first chromatin immunoprecipitation next-generation sequencing (ChIP-seq) analysis of a fungal velvet protein, providing experimental evidence that a velvet homologue in P. chrysogenum (PcVelA) acts as a direct transcriptional regulator at the DNA level in addition to functioning as a regulator at the protein level in P. chrysogenum, which was previously described. We identified many target genes that are related to processes known to be dependent on PcVelA, e.g., secondary metabolism as well as asexual and sexual development. We also identified seven PcVelA target genes that encode putative methyltransferases. Yeast two-hybrid and bimolecular fluorescence complementation analyses showed that one of the putative methyltransferases, PcLlmA, directly interacts with PcVelA. Furthermore, functional characterization of PcLlmA demonstrated that this protein is involved in the regulation of conidiosporogenesis, pellet formation, and hyphal morphology, all traits with major biotechnological relevance. IMPORTANCE Filamentous fungi are of major interest for biotechnological and pharmaceutical applications. This is due mainly to their ability to produce a wide variety of secondary metabolites, many of which are relevant as antibiotics. One of the most prominent examples is penicillin, a β-lactam antibiotic that is produced on the industrial scale by fermentation of P. chrysogenum. In recent years, the multisubunit protein complex velvet has been identified as one of the key regulators of fungal secondary metabolism and development. However, until recently, only a little has been known about how velvet mediates regulation at the molecular level. To address this issue, we performed ChIP-seq (chromatin immunoprecipitation in combination with next-generation sequencing) on and follow-up analysis of PcVelA, the core component of the velvet complex in P. chrysogenum. We demonstrate direct involvement of velvet in transcriptional control and present the putative methyltransferase PcLlmA as a new downstream factor and interaction partner of PcVelA.

scholarly journals An optimised Chromatin Immunoprecipitation (ChIP) method for starchy leaves of Nicotiana benthamiana to study histone modifications of an allotetraploid plant

2020 ◽  
Author(s):  
Buddhini Ranawaka ◽  
Milos Tanurdzic ◽  
Peter Waterhouse ◽  
Fatima Naim
Keyword(s):  
Next Generation Sequencing ◽  
Histone Modifications ◽  
Chromatin Immunoprecipitation ◽  
Nicotiana Benthamiana ◽  
Chromatin Modifications ◽  
Next Generation ◽  
High Quality ◽  
Dna Recovery ◽  
Histone 3 ◽  
Generation Sequencing

Abstract Background All flowering plants have evolved through multiple rounds of polyploidy throughout the evolutionary process. Intergenomic interactions between subgenomes in polyploid plants are predicted to induce chromatin modifications such as histone modifications to regulate expression of gene homoeologs. Nicotiana benthamiana is an ancient allotetraploid plant with ecotypes collected from climatically diverse regions of Australia. Studying the differences in chromatin landscape of this unique collection will shed light on the importance of chromatin modifications in gene regulation in polyploids as well its implications in adaptation of plants in environmentally diverse conditions. N.benthamiana is also an important biotechnological tool and it is widely used in virological research and functional genomics. Chromatin Immunoprecipitation and high throughput DNA sequencing (ChIP-seq) is well established technique used to study histone modifications. However, due to the starchy nature of mature N.benthamiana leaves, previously published protocols were unsuitable. The aim of this study was to optimise ChIP protocol for N.benthamiana leaves to facilitate comparison of chromatin modifications in two closely related ecotypes. Results Several steps of ChIP were optimised including tissue harvesting, nuclei isolation, nuclei storage, DNA shearing and DNA recovery. The higher amounts of starch in mature N.benthamiana leaves that co-precipitated with nuclei using previously published protocols, hindered chromatin shearing and resulted in low recovery of ChIP DNA. The optimised method reduced starch contamination and resulted in isolation of high quality nuclei suitable for next generation sequencing. Commonly available antibodies targeting histone 3 lysine 4 trimethylation (H3K4me3) and histone 3 lysine 9 dimethylation (H3K9me2) histone modifications were used and success of ChIP was confirmed by PCR and next generation sequencing. Conclusions An optimised ChIP method for mature leaves of N.benthamiana is described. It is relatively less laborious than previously published protocols and allows extraction of high quality nuclear genomic DNA from N.benthamiana. It is also the first comprehensive ChIP method for starchy leaves of N.benthamiana suitable for preparation of ChIP libraries for next generation sequencing.

Chromatin Immunoprecipitation of GFP-Tagged PML-Rara Coupled to High-Throughput Next Generation Sequencing.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1276-1276
Author(s):  
Nicole R. Grieselhuber ◽  
Jahangheer S. Shaik ◽  
Li-Wei Chang ◽  
Sean McGrath ◽  
Lukas D. Wartman ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Dna Binding ◽  
Fusion Protein ◽  
Chromatin Immunoprecipitation ◽  
Binding Sites ◽  
Next Generation ◽  
High Confidence ◽  
Altered Expression ◽  
Dna Binding Sites ◽  
Generation Sequencing

Abstract Abstract 1276 Poster Board I-298 The PML-RARA fusion protein produced by the t(15;17) translocation is found in acute promyelocytic leukemia (APL) and acts as an aberrant transcription factor with oncogenic properties. To define the high affinity DNA binding sites of PML-RARA, we developed a novel system based upon chromatin immunoprecipitation of eGFP tagged PML-RARA (PR), coupled to next generation sequencing. Chromatin isolated from flow sorted, GFP+ PR9 cells (isolated 24 hours after electroporation with eGFP-PR) was immunoprecipitated with a highly specific anti-GFP monoclonal antibody, and massively parallel sequencing was performed using single-end read libraries generated from both input DNA and immunoprecipitated DNA. The sequenced reads were mapped to the reference human genome using the Burrows-Wheeler Alignment tool (BWA). Using the SamTools package, alignment files were then filtered to retain only reads with phred quality scores greater than 30. Finally, Model Based Analysis of ChIP-Seq (MACS) was used to obtain the predicted binding sites (approximately 13,000 for each replicate using a p value cutoff of 0.00001). To ensure the reproducibility of called peaks, we limited our analysis to 701 sites that occurred in replicate sequencing runs of the libraries, within a tolerance of ±50 bp at the peak. Visual inspection of graphically plotted peaks further filtered this list to 421 high quality sites. Using microarray expression data for 14 APL patients and 5 flow sorted, normal promyelocyte samples, we selected neighboring genes with at least a 3-fold difference between APL and promyelocyte expression. This step yielded a list of 82 neighboring genes whose expression may be altered by PML-RARA binding to adjacent DNA. While PML-RARA is often considered a transcriptional repressor, 68% of these genes were overexpressed in APL, suggesting that PML-RARA may also function as a transcriptional activator. Furthermore, 51 genes were dysregulated in the mCG-PML-RARA murine model of APL, 16 genes demonstrated expression changes following induction of PML-RARA expression in PR9 cells, and 12 genes had altered expression in both the PR9 and murine models. Collectively, these results demonstrate an association between the high confidence putative PR binding sites and gene expression changes. We next used the sequences found within the best 89 binding sites to define 6 potential in vivo consensus sites of PML-RARA using the CONSENSUS program. Three of these predicted sites were present in greater than 40% of the 421 high confidence sites. Two of these common motifs resemble the motifs found in retinoic acid response elements (RAREs), but have less stringent conservation at the 5' end while retaining the 3' TCA sequence. Taken together, our results suggest that PML-RARA has an extended repertoire of genomic DNA binding sites compared to wild-type RARA, reflecting novel gain-of-function properties of the fusion protein. Binding of some of these sites appears to have direct consequences for the expression of several tightly linked genes, which may themselves be involved in transcriptional regulatory networks that contribute to APL pathogenesis. Disclosures No relevant conflicts of interest to declare.

Next-Generation Sequencing

2012 ◽  
Vol 42 (9) ◽  
pp. 8
Author(s):  
PETER HULICK
Keyword(s):  
Next Generation Sequencing ◽  
Next Generation ◽  
Generation Sequencing

Labordiagnostik bei gastrointestinalen Tumoren

2020 ◽  
Vol 11 (05) ◽  
pp. 232-238
Author(s):  
Marcus Kleber
Keyword(s):  
Next Generation Sequencing ◽  
Kolorektales Karzinom ◽  
Next Generation ◽  
Generation Sequencing

ZUSAMMENFASSUNGDas kolorektale Karzinom (KRK) ist einer der häufigsten malignen Tumoren in Deutschland. Einer frühzeitigen Diagnostik kommt große Bedeutung zu. Goldstandard ist hier die Koloskopie. Die aktuelle S3-Leitlinie Kolorektales Karzinom empfiehlt zum KRK-Screening den fäkalen okkulten Bluttest. Für das Monitoring von Patienten vor und nach Tumorresektion werden die Messung des Carcinoembryonalen Antigens (CEA) und der Mikrosatellitenstabilität empfohlen. Für die Auswahl der korrekten Chemotherapie scheint derzeit eine Überprüfung des Mutationsstatus, mindestens des KRAS-Gens und des BRAF-Gens, sinnvoll zu sein. Eine Reihe an neuartigen Tumormarkern befindet sich momentan in der Entwicklung, hat jedoch noch nicht die Reife für eine mögliche Anwendung in der Routinediagnostik erreicht. Den schnellsten Weg in die breite Anwendung können Next-Generation-Sequencing-basierte genetische Tests finden.

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