Chromatin Immunoprecipitation (ChIP) for Analysis of Histone Modifications and Chromatin-Associated Proteins

Condensins are evolutionarily conserved protein complexes that are required for chromosome segregation during cell division and genome organization during interphase. In Caenorhabditis elegans, a specialized condensin, which forms the core of the dosage compensation complex (DCC), binds to and represses X chromosome transcription. Here, we analyzed DCC localization and the effect of DCC depletion on histone modifications, transcription factor binding, and gene expression using chromatin immunoprecipitation sequencing and mRNA sequencing. Across the X, the DCC accumulates at accessible gene regulatory sites in active chromatin and not heterochromatin. The DCC is required for reducing the levels of activating histone modifications, including H3K4me3 and H3K27ac, but not repressive modification H3K9me3. In X-to-autosome fusion chromosomes, DCC spreading into the autosomal sequences locally reduces gene expression, thus establishing a direct link between DCC binding and repression. Together, our results indicate that DCC-mediated transcription repression is associated with a reduction in the activity of X chromosomal gene regulatory elements.

Download Full-text

Rapid and Low-Input Profiling of Histone Marks in Plants Using Nucleus CUT&Tag

Frontiers in Plant Science ◽

10.3389/fpls.2021.634679 ◽

2021 ◽

Vol 12 ◽

Author(s):

Weizhi Ouyang ◽

Xiwen Zhang ◽

Yong Peng ◽

Qing Zhang ◽

Zhilin Cao ◽

...

Keyword(s):

Histone Modifications ◽

Chromatin Immunoprecipitation ◽

Binding Sites ◽

Protein A ◽

Transcriptional Factor ◽

Experimental Procedure ◽

Histone Marks ◽

Genome Wide ◽

Efficient Alternative ◽

Tn5 Transposase

Characterizing genome-wide histone posttranscriptional modifications and transcriptional factor occupancy is crucial for deciphering their biological functions. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a powerful method for genome-wide profiling of histone modifications and transcriptional factor-binding sites. However, the current ChIP-seq experimental procedure in plants requires significant material and several days for completion. CUT&Tag is an alternative method of ChIP-seq for low-sample and single-cell epigenomic profiling using protein A-Tn5 transposase fusion proteins (PAT). In this study, we developed a nucleus CUT&Tag (nCUT&Tag) protocol based on the live-cell CUT&Tag technology. Our results indicate that nCUT&Tag could be used for histone modifications profiling in both monocot rice and dicot rapeseed using crosslinked or fresh tissues. In addition, both active and repressive histone marks such as H3K4me3 and H3K9me2 can be identified using our nCUT&Tag. More importantly, all the steps in nCUT&Tag can be finished in only 1 day, and the assay can be performed with as little as 0.01 g of plant tissue as starting materials. Therefore, our results demonstrate that nCUT&Tag is an efficient alternative strategy for plant epigenomic studies.

Download Full-text

Chromatin Immunoprecipitation Protocol for Histone Modifications and Protein–DNA Binding Analyses in Arabidopsis

Plant Epigenetics - Methods in Molecular Biology ◽

10.1007/978-1-60761-646-7_15 ◽

2010 ◽

pp. 209-220 ◽

Cited By ~ 2

Author(s):

Stéphane Pien ◽

Ueli Grossniklaus

Keyword(s):

Dna Binding ◽

Histone Modifications ◽

Chromatin Immunoprecipitation

Download Full-text

Genome-wide Analysis of Histone Modifications Distribution using the Chromatin Immunoprecipitation Sequencing Method in Magnaporthe oryzae

Journal of Visualized Experiments ◽

10.3791/62423 ◽

2021 ◽

Author(s):

Zechi Wu ◽

Wanyu Sun ◽

Sida Zhou ◽

Li Zhang ◽

Xinyu Zhao ◽

...

Keyword(s):

Histone Modifications ◽

Magnaporthe Oryzae ◽

Chromatin Immunoprecipitation ◽

Genome Wide Analysis ◽

Genome Wide ◽

Sequencing Method ◽

Chromatin Immunoprecipitation Sequencing

Download Full-text

Chromatin Immunoprecipitation for Analyzing Transcription Factor Binding and Histone Modifications in Drosophila

Methods in Molecular Biology - Drosophila ◽

10.1007/978-1-4939-6371-3_16 ◽

2016 ◽

pp. 263-277 ◽

Cited By ~ 6

Author(s):

Yad Ghavi-Helm ◽

Bingqing Zhao ◽

Eileen E. M. Furlong

Keyword(s):

Transcription Factor ◽

Histone Modifications ◽

Chromatin Immunoprecipitation ◽

Transcription Factor Binding ◽

Factor Binding

Download Full-text

An optimised chromatin immunoprecipitation (ChIP) method for starchy leaves of Nicotiana benthamiana to study histone modifications of an allotetraploid plant

Molecular Biology Reports ◽

10.1007/s11033-020-06013-1 ◽

2020 ◽

Vol 47 (12) ◽

pp. 9499-9509

Author(s):

Buddhini Ranawaka ◽

Milos Tanurdzic ◽

Peter Waterhouse ◽

Fatima Naim

Keyword(s):

Histone Modifications ◽

Chromatin Immunoprecipitation ◽

Nicotiana Benthamiana ◽

Evolutionary Process ◽

Downstream Processing ◽

Chromatin Modifications ◽

Dna Recovery ◽

Histone 3 ◽

Wide Scale ◽

Intergenomic Interactions

AbstractAll 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 chromatin landscape of this unique collection will likely 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. Generally, chromatin immunoprecipitation and high throughput DNA sequencing (ChIP-seq) is used to study chromatin modifications. However, due to the starchy nature of mature N. benthamiana leaves, previously published protocols were unsuitable. The higher amounts of starch in leaves that co-precipitated with nuclei hindered downstream processing of DNA. Here we present an optimised ChIP protocol for N. benthamiana leaves to facilitate comparison of chromatin modifications in two closely related ecotypes. Several steps of ChIP were optimised including tissue harvesting, nuclei isolation, nuclei storage, DNA shearing and DNA recovery. 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. Collectively, our optimised method is the first comprehensive ChIP method for mature starchy leaves of N. benthamiana to enable studies of chromatin landscape at the genome-wide scale.

Download Full-text

Histone modifications at the grapevine VvOMT3 locus, which encodes an enzyme responsible for methoxypyrazine production in the berry

Functional Plant Biology ◽

10.1071/fp16434 ◽

2017 ◽

Vol 44 (7) ◽

pp. 655 ◽

Cited By ~ 3

Author(s):

Juri Battilana ◽

Jake D. Dunlevy ◽

Paul K. Boss

Keyword(s):

Histone Modifications ◽

Chromatin Immunoprecipitation ◽

Developmental Regulation ◽

Grape Variety ◽

Histone Tail ◽

Dicarbonyl Compound ◽

Grape Berries ◽

Allelic Differences ◽

Chromatin Arrangement ◽

Histone Tail Modification

Some herbaceous characters in wine are attributed to the presence of aroma compounds collectively known as methoxypyrazines (MPs). In grape berries their formation has been hypothesised to start from a reaction of two amino acids or an amino acid and an unknown 1,2-dicarbonyl compound, leading to the formation of hydroxypyrazine, which is then enzymatically methylated to form a MP. The enzyme responsible of the formation of 3-isobutyl-2-methoxypyrazine has been recently identified as VvOMT3 whose regulation is still not understood. The concentration of MPs in grapes is known to be influenced by development, environmental stimuli and most importantly grape variety. In order to investigate the chromatin arrangement of that region a chromatin immunoprecipitation analysis has been performed and putative differences in epigenetic regulation of VvOMT3 spatially between the skin and flesh tissues and also temporally during fruit development have been detected. There are also allelic differences in VvOMT3 histone modifications which are maintained in subsequent generations. This study provides evidence of histone tail modification of the VvOMT3 locus in grapevine, which may play a role in the spatial and developmental regulation of the expression of this gene.

Download Full-text

The nucleoid-associated protein Gbn binds to GATC sequences and affects sporulation and antibiotic production in Streptomyces

10.1101/2021.02.06.430045 ◽

2021 ◽

Author(s):

Chao Du ◽

Joost Willemse ◽

Amanda M. Erkelens ◽

Victor J. Carrion ◽

Remus T. Dame ◽

...

Keyword(s):

Dna Binding ◽

Chromatin Immunoprecipitation ◽

Antibiotic Production ◽

Consensus Sequence ◽

Associated Proteins ◽

Chromatin Immunoprecipitation Sequencing ◽

Number Of Binding Sites ◽

Trna Editing ◽

Editing Enzyme

ABSTRACTBacterial chromosome structure is organized by a diverse group of proteins collectively called nucleoid-associated proteins (NAPs). Many NAPs have been studied in detail in Streptomyces, including Lsr2, HupA, HupS, and sIHF. Here, we show that SCO1839 represents a novel family of small NAPs unique to Actinobacteria and recognizes a consensus sequence consisting of GATC followed by (A/T)T. The protein was designated Gbn for GATC-binding NAP. Chromatin immunoprecipitation sequencing (ChIP-Seq) detected more than 2800 binding regions, encompassing some 3600 GATCWT motifs, which comprise 55% of all motifs in the S. coelicolor genome. DNA binding of Gbn in vitro increased DNA stiffness but not compaction, suggesting a role in regulation rather than chromosome organization. Despite the huge number of binding sites, the DNA binding profiles were nearly identical during vegetative and aerial growth. The exceptions were SCO1311 and SCOt32, for a tRNA editing enzyme and a tRNA that recognises the rare leucine codon CUA, respectively, which were nearly exclusively bound during vegetative growth. Deletion of gbn led to pleiotropic alterations in developmental timing, morphogenesis and antibiotic production. Taken together, our data show that Gbn is a highly pleiotropic NAP that impacts growth and development in streptomycetes.

Download Full-text

Proteomic characterization of chromosomal common fragile site (CFS)-associated proteins uncovers ATRX as a regulator of CFS stability

Nucleic Acids Research ◽

10.1093/nar/gkz510 ◽

2019 ◽

Vol 47 (15) ◽

pp. 8004-8018 ◽

Cited By ~ 8

Author(s):

David Pladevall-Morera ◽

Stephanie Munk ◽

Andreas Ingham ◽

Lorenza Garribba ◽

Eliene Albers ◽

...

Keyword(s):

Chromatin Immunoprecipitation ◽

Fragile Site ◽

Global Analysis ◽

Fragile Sites ◽

Replication Proteins ◽

Chromosomal Breaks ◽

Associated Proteins ◽

Genomic Regions ◽

Conserved Genomic Regions

Abstract Common fragile sites (CFSs) are conserved genomic regions prone to break under conditions of replication stress (RS). Thus, CFSs are hotspots for rearrangements in cancer and contribute to its chromosomal instability. Here, we have performed a global analysis of proteins that recruit to CFSs upon mild RS to identify novel players in CFS stability. To this end, we performed Chromatin Immunoprecipitation (ChIP) of FANCD2, a protein that localizes specifically to CFSs in G2/M, coupled to mass spectrometry to acquire a CFS interactome. Our strategy was validated by the enrichment of many known regulators of CFS maintenance, including Fanconi Anemia, DNA repair and replication proteins. Among the proteins identified with unknown functions at CFSs was the chromatin remodeler ATRX. Here we demonstrate that ATRX forms foci at a fraction of CFSs upon RS, and that ATRX depletion increases the occurrence of chromosomal breaks, a phenotype further exacerbated under mild RS conditions. Accordingly, ATRX depletion increases the number of 53BP1 bodies and micronuclei, overall indicating that ATRX is required for CFS stability. Overall, our study provides the first proteomic characterization of CFSs as a valuable resource for the identification of novel regulators of CFS stability.

Download Full-text

A Computational Model of Quantitative Chromatin Immunoprecipitation (ChIP) Analysis

Cancer Informatics ◽

10.4137/cin.s295 ◽

2008 ◽

Vol 6 ◽

pp. CIN.S295 ◽

Cited By ~ 1

Author(s):

Jingping Xie ◽

Philip S. Crooke ◽

Brett A. McKinney ◽

Joel Soltman ◽

Stephen J. Brandt

Keyword(s):

Computational Model ◽

Chromatin Immunoprecipitation ◽

Pcr Primers ◽

Enzymatic Digestion ◽

Cross Linking ◽

Associated Proteins ◽

Width Distribution ◽

The Mean ◽

Chemical Cross Linking ◽

Chip Analysis

Chromatin immunoprecipitation (ChIP) analysis is widely used to identify the locations in genomes occupied by transcription factors (TFs). The approach involves chemical cross-linking of DNA with associated proteins, fragmentation of chromatin by sonication or enzymatic digestion, immunoprecipitation of the fragments containing the protein of interest, and then PCR or hybridization analysis to characterize and quantify the genomic sequences enriched. We developed a computational model of quantitative ChIP analysis to elucidate the factors contributing to the method's resolution. The most important variables identified by the model were, in order of importance, the spacing of the PCR primers, the mean length of the chromatin fragments, and, unexpectedly, the type of fragment width distribution, with very small DNA fragments and smaller amplicons providing the best resolution of TF binding. One of the major predictions of the model was also validated experimentally.

Download Full-text