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Development and validation of μChIP. (A) Genomic regions examined. Bars and numbers indicate the position of ChIP amplicons relative to the transcription start site (TSS). PE, PP and CR1 delineate the proximal enhancer, proximal promoter and conserved region 1 of OCT4, respectively. (B) RT-PCR analysis of expression of GAPDH, OCT4, NANOG and SLC10A6 in NCCIT cells. Plus (+) and minus (−) indicate the presence or absence of reverse transcriptase (RT). (C–E) ChIP analysis of H3 modifications (x-axis) in NCCIT cells on promoters of indicated genes. (C) Analysis from chromatin of 100 000 cells, (D) 1000 cells and (E) 100 cells. (F–H) ChIP analysis of RNAPII binding to GAPDH, OCT4 and SLC10A6 promoters in the same chromatin samples as those examined for histones in (C–E). In (C–H), data are expressed as percent precipitation relative to input chromatin (mean ± SD; 2–9 independent experiments).
Source publication
Chromatin immunoprecipitation (ChIP) is a powerful technique for studying protein–DNA interactions. Drawbacks of current ChIP
assays however are a requirement for large cell numbers, which limits applicability of ChIP to rare cell samples, and/or lengthy
procedures with limited applications. There are to date no protocols for fast and parallel ChIP...
Contexts in source publication
Context 1
... test and validate the mChIP assay, we monitored the association of H3K9ac (a modification indicative of transcriptional activity), H3K4m2 and H3K4m3 [modifi- cations associated with promoters but not necessarily indicative or predictive of transcriptional activity (16,17)] and of H3K9m2, H3K9m3 and H3K27m3 (three transcriptionally repressive marks) with the GAPDH, OCT4 and NANOG promoters in NCCIT cells ( Figure 1A). RT-PCR analysis shows that GAPDH, OCT4 and NANOG were expressed ( Figure 1B). ...Context 2
... test and validate the mChIP assay, we monitored the association of H3K9ac (a modification indicative of transcriptional activity), H3K4m2 and H3K4m3 [modifi- cations associated with promoters but not necessarily indicative or predictive of transcriptional activity (16,17)] and of H3K9m2, H3K9m3 and H3K27m3 (three transcriptionally repressive marks) with the GAPDH, OCT4 and NANOG promoters in NCCIT cells ( Figure 1A). RT-PCR analysis shows that GAPDH, OCT4 and NANOG were expressed ( Figure 1B). Starting material was for each ChIP, either chromatin from 100 000 cells, chromatin from $1000 cells prepared from a starting 10 000 cells and divided into nine samples ('1000-cell ChIP'), or chromatin from $100 cells prepared from 1000 cells and divided into nine samples ('100-cell ChIP'). ...Context 3
... PCR analysis of the ChIP DNA shows that with chromatin from 100 000 cells, H3K9ac, H3K4m2 and H3K4m3 were enriched on all promoters, whereas repressive histone marks were at background level ( Figure 1C). This was in agreement with activation of the genes in NCCIT cells ( Figure 1B) and with previously published Q 2 ChIP data (12). ...Context 4
... PCR analysis of the ChIP DNA shows that with chromatin from 100 000 cells, H3K9ac, H3K4m2 and H3K4m3 were enriched on all promoters, whereas repressive histone marks were at background level ( Figure 1C). This was in agreement with activation of the genes in NCCIT cells ( Figure 1B) and with previously published Q 2 ChIP data (12). Remarkably, the 1000-cell ChIP ( Figure 1D) and the 100-cell ChIP assays ( Figure 1E) produced a similar histone enrichment profile as with 100 000 cells, indicating that scaling down by 1000- fold maintained specificity of the assay. ...Context 5
... was in agreement with activation of the genes in NCCIT cells ( Figure 1B) and with previously published Q 2 ChIP data (12). Remarkably, the 1000-cell ChIP ( Figure 1D) and the 100-cell ChIP assays ( Figure 1E) produced a similar histone enrichment profile as with 100 000 cells, indicating that scaling down by 1000- fold maintained specificity of the assay. ...Context 6
... was in agreement with activation of the genes in NCCIT cells ( Figure 1B) and with previously published Q 2 ChIP data (12). Remarkably, the 1000-cell ChIP ( Figure 1D) and the 100-cell ChIP assays ( Figure 1E) produced a similar histone enrichment profile as with 100 000 cells, indicating that scaling down by 1000- fold maintained specificity of the assay. ...Context 7
... consistently noted however, enhanced precipitation, relative to input, from small chromatin samples (compare y-axis scales in Figure 1C-E). This is suggested to be due to an increased antibody/bead-to-antigen ratio. ...Context 8
... is suggested to be due to an increased antibody/bead-to-antigen ratio. Indeed, we showed that reducing antibody concentration to 40% relative to a standard Q 2 ChIP condition reduced the PCR signal by $50% while a 4-fold increase in antibody concentration enhanced the PCR signal by >30% (Supplementary Data, Figure 1). Nevertheless, while the ratio of antibody-to-chromatin increases 1000-fold between the 100 000-cell and the 100-cell ChIP assay, the ChIP efficiency (precipitation relative to input) increased by 1.5-2-fold. ...Context 9
... indicates that the relationship between antibody concentration and ChIP efficiency is non-linear in the range examined and that with low amounts of chromatin, the antibody concentration is not limiting. The relative background signal also increased when scaling down ChIP ( Figure 1D and E); however this did not affect the overall interpretation of the results (see Discussion). These results indicate therefore that mChIP maintains ...Context 10
... of RNAPII to target promoters has been used as an indicator of transcriptional activity or potential for activity in genome-wide epigenetic studies (3,18). RNAPII ChIPs were carried out in parallel to histone ChIPs presented before, from the same chromatin preparations ( Figure 1F-H). RNAPII association with GAPDH and OCT4 promoters was clearly detected under each condition. ...Context 11
... association with GAPDH and OCT4 promoters was clearly detected under each condition. As negative control for RNAPII binding, we monitored RNAPII association with the inactive SLC10A6 promoter ( Figure 1B). Binding was dramati- cally weaker than with GAPDH and OCT4 and con- sistently near background levels ( Figure 1F-H). ...Context 12
... negative control for RNAPII binding, we monitored RNAPII association with the inactive SLC10A6 promoter ( Figure 1B). Binding was dramati- cally weaker than with GAPDH and OCT4 and con- sistently near background levels ( Figure 1F-H). We concluded that mChIP is suited for analysis of histone and RNAPII binding to genomic loci in parallel immunoprecipitations from chromatin from as few as 100 cells. ...Context 13
... DNA isolation also enhances DNA recovery (data not shown). Figure 2A shows that profiles of H3K9ac, H3K4m2 and H3K9m2 association with GAPDH, OCT4 and NANOG promoters in these samples were remarkably similar to those produced from those of a split chromatin sample from 1000 cells (compare with Figure 1E). In addition, mChIP enabled the detection of H3K9m2 enrichment on the SLC10A6 promoter, in agreement with its inactive state in NCCIT cells (Figure 2A). ...Context 14
... over H3K9m2 decreases by $5-fold relative to 100 000-cell ChIP when the data are presented includ- ing background (compare, e.g. Figure 1C with E). Nevertheless, all modified histone binding profiles on the loci examined are maintained under each condition: H3K9 acetylated promoters remain clearly marked as acetylated, with no (or background) levels of H3K9 and H3K27 methylation. ...Context 15
... is likely that reducing cell numbers in ChIP assays systematically results in enhanced background [see also (12)], an issue simply dealt with recently in a miniChIP assay by subtracting background values (26). Clearly, a background subtraction in Figures 1 and 2 in the present article would produce histone enrichment profiles identical to those of 100 000-cell ChIP assays; however, we favor in this communication the presentation of all data to emphasize awareness of the background issue. Biological interpretation of the data remains unaffected. ...Citations
... ChIP on the sorted progenitors and b-cells was carried out according to a low-cell number micro-ChIP protocol, with minor modifications (Dahl and Collas 2008;Dhawan et al. 2015). The antibodies and primers used for ChIP are listed in the Supplemental Tables 3 and 4. Bisulfite conversion of DNA from sorted cells and islets and sequencing of the converted DNA was performed according to established methods (Millar et al. 2002;Dhawan et al. 2011) using primers described in Supplemental Table 5. ...
The molecular and functional heterogeneity of pancreatic β -cells is well recognized. Pancreatic islets harbor a small subset of β -cells that co-express Tyrosine Hydroxylase (TH), an enzyme involved in synthesis of catecholamines that repress insulin secretion. Restriction of this sub-population within islets is essential for appropriate insulin secretion. However, the distinguishing characteristics of this subpopulation and the mechanisms that restrict TH expression in β -cells are not known. Here, we define the specific molecular and metabolic characteristics of the TH+ β -cells and show that TH expression in β -cells is restricted by DNA methylation patterning during β -cell lineage specification. Ablation of de novo DNA methyltransferase Dnmt3a in the pancreatic- and endocrine-progenitor lineages results in a dramatic increase in the proportion of TH+ β -cells, while β -cell specific ablation of Dnmt3a has no effect on this sub-population. We demonstrate that maintenance of Th promoter DNA methylation patterns is essential for its continued restriction in postnatal β -cells, and that loss of DNA methylation dysregulates TH expression in β -cells in response to chronic overnutrition, contributing to impairment of β -cell identity. These data highlight the essential requirement of DNA methylation patterning in regulating endocrine cell fates, and reveal a novel role of DNA methylation in β -cell heterogeneity.
... Also, microChIP (μChIP) assay is another improved ChIP-based method, which is appropriate for up to nine parallel ChIPs of modified histone of 1,000 cells. The assay is applied to genome-wide studies and analysis of multiple epigenetic modifications, too [81,102,103]. On the other hand, the Fast-ChIP assay has improved the basic ChIP assay in two steps, and this improvement accelerates the protocol significantly and helps to detect different epigenetic factors [104,105] in more than one site or even through the whole genome. ...
Epigenetics refers to nucleotide sequence-independent events, and heritable changes, including DNA methylation and histone modification (as the two main processes), contributing to the phenotypic features of the cell. Both genetics and epigenetics contribute to determining the outcome of regulatory gene expression systems. Indeed, the flexibility of epigenetic effects and stability of genetic coding lead to gene regulation complexity in response signals. Since some epigenetic changes are significant in abnormalities such as cancers and neurodegenerative diseases, the initial changes, dynamic and reversible properties, and diagnostic potential of epigenomic phenomena are subject to epigenome-wide association studies (EWAS) for therapeutic aims. Based on recent studies, methodological developments are necessary to improve epigenetic research. As a result, several methods have been developed to explore epigenetic alterations at low, medium, and high scales, focusing on DNA methylation and histone modification detection. In this research field, bisulfite-, enzyme sensitivity- and antibody specificity-based techniques are used for DNA methylation, whereas histone modifications are gained based on antibody recognition. This review provides a mechanism-based understanding and comparative overview of the most common techniques for detecting the status of epigenetic effects, including DNA methylation and histone modifications, for applicable approaches from low- to high-throughput scales.
... While ChIP is valuable, the strategy can detect and analyze just a single histone modification at a time. Traditional strategies to investigate chromatin are restricted in their capability to give enough data at a single cell level data for uncommon cell or cell differentiation research [13,53,73]. ...
Epigenetic inheritance occurs due to different mechanisms such as chromatin and histone modifications, DNA methylation and processes mediated by non-coding RNAs. It leads to changes in gene expressions and the emergence of new traits in different organisms in many diseases such as cancer. Recent advances in experimental methods led to the identification of epigenetic target sites in various organisms. Computational approaches have enabled us to analyze mass data produced by these methods. Next-generation sequencing (NGS) methods have been broadly used to identify these target sites and their patterns. By using these patterns, the emergence of diseases could be prognosticated. In this study, target site prediction tools for two major epigenetic mechanisms comprising histone modification and DNA methylation are reviewed. Publicly accessible databases are reviewed as well. Some suggestions regarding the state-of-the-art methods and databases have been made, including examining patterns of epigenetic changes that are important in epigenotypes detection.
... To facilitate ChIP-Seq profiling of low input samples, a range of strategies have been developed (see Fig. S1 for a selection of main strategies (O'Neill et al. 2006;Dahl and Collas 2008b;Dahl and Collas 2008a;Adli and Bernstein 2011;Brind'Amour et al. 2015;Rotem et al. 2015;Schmidl et al. 2015;Dahl et al. 2016;van Galen et al. 2016;Weiner et al. 2016;Zhang et al. 2016;Skene et al. 2018;Ai et al. 2019;Kaya-Okur et al. 2019)). Methods that have been applied include barcoding and pooling of multiple samples in the ChIP reaction (Rotem et al. 2015;van Galen et al. 2016;Weiner et al. 2016), small volume sonication (Adli and Bernstein 2011), substitution of sonication by a native MNase digestion approach (Brind'Amour et al. 2015), the use of carrier material (mainly used for ChIP-qPCR) (O'Neill et al. 2006) and application of a transposase for DNA cleavage and library generation (Schmidl et al. 2015;Ai et al. 2019;Kaya-Okur et al. 2019). ...
... Because traditional ChIP-Seq approaches require large amounts of material (Ho et al. 2011;Chen et al. 2012;Landt et al. 2012), a range of previous studies such as have worked towards procedures to downscale the ChIP procedure (Fig S1 (O 'Neill et al. 2006;Dahl and Collas 2008b;Dahl and Collas 2008a;Adli and Bernstein 2011;Brind'Amour et al. 2015;Rotem et al. 2015;Schmidl et . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity. ...
Epigenetic profiling by ChIP-Seq has become a powerful tool for genome-wide identification of regulatory elements, for defining transcriptional regulatory networks and for screening for biomarkers. However, the ChIP-Seq protocol for low-input samples is laborious, time-consuming and suffers from experimental variation, resulting in poor reproducibility and low throughput. Although prototypic microfluidic ChIP-Seq platforms have been developed, these are poorly transferable as they require sophisticated custom-made equipment and in-depth microfluidic and ChIP expertise, while lacking parallelisation. To enable standardized, automated ChIP-Seq profiling of low-input samples, we constructed PDMS-based plates containing microfluidic Integrated Fluidic Circuits capable of performing 24 sensitive ChIP reactions within 30 minutes hands-on time. These disposable plates can conveniently be loaded into a widely available controller for pneumatics and thermocycling, making the ChIP-Seq procedure Plug and Play (PnP). We demonstrate high-quality ChIP-seq on hundreds to few thousands of cells for multiple widely-profiled post-translational histone modifications, together allowing genome-wide identification of regulatory elements. As proof of principle, we managed to generate high-quality epigenetic profiles of rare totipotent subpopulations of mESCs using our platform. In light of the ready-to-go ChIP plates and the automated workflow, we named our procedure PnP-ChIP-Seq. PnP-ChIP-Seq allows non-expert labs worldwide to conveniently run robust, standardized ChIP-Seq, while its high-throughput, consistency and sensitivity paves the way towards large-scale profiling of precious sample types such as rare subpopulations of cells or biopsies.
Reviewer link to data
All sequencing data has been submitted to the NCBI GEO database. Reviewer link: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=klwnocicrpaxrkv&acc=GSE120673
... A challenge with the ChIP method has long been the need for a large amount of input material, meaning that its application to low--cell--number samples has been limited. However, because many interesting biological cell types are difficult to obtain in high numbers the continuous scaling down of ChIP protocols has been an ongoing effort starting with the pioneering of low--cell--number native ChIP and cross--linked ChIP more than a decade ago: The scaling down of single--locus ChIP (Dahl & Collas, 2007;O'Neill, VerMilyea, & Turner, 2006), ChIP--chip and ChIP--seq has allowed for analysis of cell types only available in limited numbers, such as oocytes and embryos (Brind'Amour et al., 2015;Dahl et al., 2016;Dahl, Reiner, Klungland, Wakayama, & Collas, 2010;Xiaoyu Liu et al., 2016b;Zhang et al., 2016), primordial germ cells (Sachs et al. 2013;Ng et al. 2013), sorted homogenous rare--cell populations (Adli, Zhu, & Bernstein, 2010;Jakobsen et al., 2015;Lara--Astiaso et al., 2014;van Galen et al., 2016) and tissue biopsies (Dahl & Collas, 2008b;Zwart et al., 2013). These in vivo derived cell types are superior to their in vitro counterparts in gaining biological insights that are valid in the context of an organism. ...
... Each 100--cell ChIP contained sufficient ChIP DNA to allow for analysis of three loci by duplicate qPCR. This protocol has been validated using both human pluripotent embryonal carcinoma cells and human solid tumor biopsy material by assessing posttranslational modifications of histone 3 (H3) as well as RNA polymerase ΙΙ (RNA Pol ΙΙ) occupancy at developmentally regulated genes (Dahl & Collas, 2008b). A drawback with ChIP assays targeting only a few loci, is that these strategies necessitate some prior knowledge about which regions are likely to be bound by the factor or modified histone in question. ...
Chromatin immunoprecipitation (ChIP) enables mapping of specific histone modifications or chromatin‐associated factors in the genome and represents a powerful tool in the study of chromatin and genome regulation. Importantly, recent technological advances that couple ChIP with whole‐genome high‐throughput sequencing (ChIP‐seq) now allow the mapping of chromatin factors throughout the genome. However, the requirement for large amounts of ChIP‐seq input material has long made it challenging to assess chromatin profiles of cell types only available in limited numbers. For many cell types, it is not feasible to reach high numbers when collecting them as homogeneous cell populations in vivo. Nonetheless, it is an advantage to work with pure cell populations to reach robust biological conclusions. Here, we review (a) how ChIP protocols have been scaled down for use with as little as a few hundred cells; (b) which considerations to be aware of when preparing small‐scale ChIP‐seq and analyzing data; and (c) the potential of small‐scale ChIP‐seq datasets for elucidating chromatin dynamics in various biological systems, including some examples such as oocyte maturation and preimplantation embryo development.
This article is categorized under: Laboratory Methods and Technologies > Genetic/Genomic Methods
Developmental Biology > Developmental Processes in Health and Disease
Biological Mechanisms > Cell Fates
... Chromatin immunoprecipitation (ChIP) experiments with purified aand b-cells were carried out using the micro-ChIP protocol (20). Islets from 2-month-old mice were used for MafB (Bethyl Laboratories) and Dnmt3a (Novus Biologicals) ChIP. ...
The sustained expression of the MAFB transcription factor in human islet β-cells represents a distinct difference in mice. Moreover, mRNA expression of closely related and islet β-cell-enriched MAFA does not peak in humans until after 9 years of age. We show that the MAFA protein also is weakly produced within the juvenile human islet β-cell population and that MafB expression is postnatally restricted in mouse β-cells by de novo DNA methylation. To gain insight into how MAFB affects human β-cells, we developed a mouse model to ectopically express MafB in adult mouse β-cells using MafA transcriptional control sequences. Coexpression of MafB with MafA had no overt impact on mouse β-cells, suggesting that the human adult β-cell MAFA/MAFB heterodimer is functionally equivalent to the mouse MafA homodimer. However, MafB alone was unable to rescue the islet β-cell defects in a mouse mutant lacking MafA in β-cells. Of note, transgenic production of MafB in β-cells elevated tryptophan hydroxylase 1 mRNA production during pregnancy, which drives the serotonin biosynthesis critical for adaptive maternal β-cell responses. Together, these studies provide novel insight into the role of MAFB in human islet β-cells.
... Chromatin immunoprecipitation (ChIP) has become a technique of choice to dissect the composition of transcriptional regulators in tissue culture cells and in primary tissue [7][8][9]. Therefore, the applications of ChIP to mouse embryonic tissue provide numerous possibilities to dissect regulatory networks at specific stages and at in vivo contexts that otherwise are difficult to be addressed in tissue culture systems. This, in combination with deep-sequencing methodologies increases the potential and the informative power of ChIP-based methods. ...
Epigenetic regulation is achieved at many levels by different factors such as tissue-specific transcription factors, members of the basal transcriptional apparatus, chromatin-binding proteins, and noncoding RNAs. Importantly, chromatin structure dictates the availability of a specific genomic locus for transcriptional activation as well as the efficiency with which transcription can occur. Chromatin immunoprecipitation (ChIP) is a method that allows elucidating gene regulation at the molecular level by assessing if chromatin modifications or proteins are present at a specific locus. Initially, the majority of ChIP experiments were performed on cultured cell lines and more recently this technique has been adapted to a variety of tissues in different model organisms. Using ChIP on mouse embryos, it is possible to document the presence or absence of specific proteins and chromatin modifications at genomic loci in vivo during mammalian development and to get biological meaning from observations made on tissue culture analyses. We describe here a ChIP protocol on freshly isolated mouse embryonic somites for in vivo analysis of muscle specific transcription factor binding on chromatin. This protocol has been easily adapted to other mouse embryonic tissues and has also been successfully scaled up to perform ChIP-Seq.
... Furthermore, during the last decade, the basic ChIP method has been developed to more specifically answer different research questions and to utilize more recent analysis technologies, so diverse ChIP methods have been described in the literature. Such are the mChIP-seq for low, micro-scale sample amount analysis, and the modern single-molecule real-time sequencing (SMRT; "third generation sequencing") of ChIP-samples (Dahl and Collas, 2008;Wu et al., 2016). ...
Disease development is influenced by both environmental conditions and genetic factors. This is the case also for cardiovascular diseases, where genetic variation in DNA sequence (risk alleles, single nucleotide polymorphisms) can increase the risk of the disease, but environmental factors (nutrition, stress, exercise) can trigger disease manifestations. Epigenetic factors can respond to human environment and habits and affect the expression of genes. Epigenetic marks can also be inherited to the offspring, as observed in the studies of famine. Thus, besides the DNA sequence itself, the epigenetic status of tissues need to be taken into account in the pathogenesis of diseases and their treatment. This article describes the most common epigenetic factors playing a role in gene regulation, namely DNA methylation, histone modifications, and RNA transcription. Current tools for studying epigenomics are also discussed.
... Such modifications include: (i) low-volume, low-binding (e.g. siliconized) plasticware and minimized exposure to plastic surfaces [15,16]. (ii) Faster sample handling and the merging of procedures, particularly during elution, high-temperature de-crosslinking and proteinase digestion, which may reduce sample damage and loss [12]. ...
In the past decade, chromatin immunoprecipitation sequencing (ChIP-seq) has emerged as the dominant technique for those wishing to perform genome-wide protein:DNA profiling. Owing to the tissue- and cell-type-specific nature of epigenetic marks, the field has been driven towards obtaining data from ever-lower cell numbers. In this review, we focus on the methodological developments that have lowered input requirements and the biological findings they have enabled, as we strive towards the ultimate goal of robust single-cell ChIP-seq.
... After sonication, the sample was centrifuged at 16,000 g for 15 min at 4˚C, the chromatin-containing supernatant transferred to a fresh microfuge tube and 70 μl RIPA Buffer (36.7 mM Tris.HCl pH 8, 2.5 mM EDTA.Na 2 , 0.01% SDS, 2.46% Triton X-100, 374 mM NaCl) containing protease inhibitors added to the chromatin sample. The ChIP reaction, washes and DNA purification were performed as in Dahl and Collas [56,57]. In brief, magnetic beads were coated with 2.4 μg of rabbit anti-H3K27me3 antibody (Millipore 07-449) and incubated overnight in a volume of 100 μl with chromatin from~100,000 YFP + sorted cells. ...
It is now well established that eukaryote genomes have a common architectural organization into topologically associated domains (TADs) and evidence is accumulating that this organization plays an important role in gene regulation. However, the mechanisms that partition the genome into TADs and the nature of domain boundaries are still poorly understood. We have investigated boundary regions in the Drosophila genome and find that they can be identified as domains of very low H3K27me3. The genome-wide H3K27me3 profile partitions into two states; very low H3K27me3 identifies Depleted (D) domains that contain housekeeping genes and their regulators such as the histone acetyltransferase-containing NSL complex, whereas domains containing moderate-to-high levels of H3K27me3 (Enriched or E domains) are associated with regulated genes, irrespective of whether they are active or inactive. The D domains correlate with the boundaries of TADs and are enriched in a subset of architectural proteins, particularly Chromator, BEAF-32, and Z4/Putzig. However, rather than being clustered at the borders of these domains, these proteins bind throughout the H3K27me3-depleted regions and are much more strongly associated with the transcription start sites of housekeeping genes than with the H3K27me3 domain boundaries. While we have not demonstrated causality, we suggest that the D domain chromatin state, characterised by very low or absent H3K27me3 and established by housekeeping gene regulators, acts to separate topological domains thereby setting up the domain architecture of the genome.
