scholarly journals High-Resolution Chromatin Immunoprecipitation: ChIP-Sequencing

2017 ◽  
pp. 61-73 ◽  
Author(s):  
Roxanne E. Diaz ◽  
Aurore Sanchez ◽  
Véronique Anton Le Berre ◽  
Jean-Yves Bouet
Keyword(s):  
High Resolution ◽  
Chromatin Immunoprecipitation ◽  
Chip Sequencing

High-Resolution Chromatin Immunoprecipitation (ChIP) Sequencing Identifies Novel Binding Targets and Prognostic Role for SOX11 in Mantle Cell Lymphoma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 585-585
Author(s):  
Pei-Yu Kuo ◽  
Violetta V. Leshchenko ◽  
Tobias A. Gellen ◽  
Netha Ulahannan ◽  
Paul Zumbo ◽  
...  
Keyword(s):  
High Resolution ◽  
Cell Lines ◽  
Chromatin Immunoprecipitation ◽  
Target Genes ◽  
Prognostic Impact ◽  
Chip Sequencing ◽  
Mantle Cell ◽  
Sox11 Expression ◽  
Direct Binding

Abstract Abstract 585 SOX11 belongs to the high-mobility group of transcription factors that bind to DNA and induce large conformational changes that facilitate the binding of other transcription factors. Genome-wide methylation analysis of Mantle cell lymphoma (MCL) patients using Nimblegen arrays identified SOX11 as hypomethylated and overexpressed in comparison to purified naïve B cells, which are the cells from which MCLs originate. Direct sequencing of SOX11 in MCL cell lines and patient samples failed to identify any activating mutations in SOX11, strengthening the role of aberrant promoter hypomethylation in MCL as the cause of its overexpression. SOX11 expression has been previously compared between Non-Hodgkin's lymphomas and found to be specific for MCL. The function and direct binding targets of SOX11 are unknown. To understand the functional role of SOX11 in MCL, we used high-resolution whole-genome chromatin immunoprecipitation sequencing (ChIP-Seq) to identify the target genes directly bound by SOX11. As a model, genomic DNA from the Granta-519 MCL cell line was immunoprecipitated by SOX11 antibody and IgG and genomic enrichment sites were analyzed by Illumina sequencing. Using stringent statistical criteria (p-value<0.01, fold change≥1.5, and FDR<0.01) we identified 5682 peak positions with significant enrichment of ChIP sequencing reads corresponding to 888 unique genes. Sequencing runs from independent immunoprecipitates had more than 95% overlap in binding targets. These targets were filtered with published gene sets of differentially expressed genes following SOX11 overexpression in MCL cell lines. Pathway analysis revealed cell cycle, FOXm transcriptional network, and TGF-beta receptor signaling as the top three significantly enriched pathways by SOX11 target genes. All three pathways prominently consist of genes with anti-proliferative effects in MCL and B-NHL in general. To test our hypothesis, we transfected SOX11 by lentiviral plasmids into four MCL cell lines with varying levels of intrinsic SOX11 expression. Overexpression of SOX11 led to decreased proliferation in all four cell lines as measured by long-term tracing of living cells using a fluorescent labeled dye to track cell division. Ingenuity Pathway Analysis also identified the tumor promoting Wnt, PKC and IGFR pathways as being significantly enriched by the binding targets. We validated direct binding of SOX11 to RB1, Wnt4 and PKCA by quantitive PCR of ChIP products from Granta-519 cells. RB1 is a key regulator of entry into cell division tumor suppressor in MCL whereas Wnt signaling activates c-myc, which is oncogenic in MCL. Previously published studies have indicated a significant negative prognostic impact of SOX11 expression on overall survival in MCL. We therefore analyzed SOX11 expression in two large independent tissue microarrays from University of Wisconsin (UW) and British Columbia Cancer Agency (BCCA), respectively. MCL patient samples were scored by experienced hematopathologists. High SOX11 expression was associated with better survival: 41 months for SOX11 positive vs. 11 months for SOX11 negative, (p=0.05) in 58 patients in the UW dataset, however the larger BCCA dataset of 140 patients did not show any statistically significant impact of SOX11 expression on survival (p=0.15). These findings suggest that the prognostic impact of SOX11 is heterogenous, and a subset of patients with high SOX11 expression may have improved survival. In conclusion, high-resolution ChIP-Seq identifies more than 800 novel SOX11 direct binding targets. Differential activation of the diverse biological pathways affected by SOX11 target genes may help explain the differential impact of SOX11 expression on patient outcomes. Disclosures: No relevant conflicts of interest to declare.

scholarly journals A strand specific high resolution normalization method for chip-sequencing data employing multiple experimental control measurements

2012 ◽  
Vol 7 (1) ◽  
pp. 2 ◽  
Author(s):  
Stefan Enroth ◽  
Claes R Andersson ◽  
Robin Andersson ◽  
Claes Wadelius ◽  
Mats G Gustafsson ◽  
...  
Keyword(s):  
High Resolution ◽  
Normalization Method ◽  
Sequencing Data ◽  
Experimental Control ◽  
Chip Sequencing

scholarly journals Antibody performance in ChIP-sequencing assays: From quality scores of public data sets to quantitative certification

F1000Research ◽  
2016 ◽  
Vol 5 ◽  
pp. 54 ◽  
Author(s):  
Marco-Antonio Mendoza-Parra ◽  
Vincent Saravaki ◽  
Pierre-Etienne Cholley ◽  
Matthias Blum ◽  
Benjamin Billoré ◽  
...  
Keyword(s):  
Chromatin Immunoprecipitation ◽  
Polyclonal Antibodies ◽  
Present Report ◽  
Data Sets ◽  
Chip Sequencing ◽  
Public Data ◽  
Original Genome ◽  
Commercial Source ◽  
Attribute Quality

We have established a certification system for antibodies to be used in chromatin immunoprecipitation assays coupled to massive parallel sequencing (ChIP-seq). This certification comprises a standardized ChIP procedure and the attribution of a numerical quality control indicator (QCi) to biological replicate experiments. The QCi computation is based on a universally applicable quality assessment that quantitates the global deviation of randomly sampled subsets of ChIP-seq dataset with the original genome-aligned sequence reads. Comparison with a QCi database for >28,000 ChIP-seq assays were used to attribute quality grades (ranging from ‘AAA’ to ‘DDD’) to a given dataset. In the present report we used the numerical QC system to assess the factors influencing the quality of ChIP-seq assays, including the nature of the target, the sequencing depth and the commercial source of the antibody.  We have used this approach specifically to certify mono and polyclonal antibodies obtained from Active Motif directed against the histone modification marks H3K4me3, H3K27ac and H3K9ac for ChIP-seq. The antibodies received the grades AAA to BBC (www.ngs-qc.org). We propose to attribute such quantitative grading of all antibodies attributed with the label “ChIP-seq grade”.

scholarly journals High-resolution, genome-wide mapping of positive supercoiling in chromosomes

2021 ◽  
Author(s):  
Monica S. Guo ◽  
Ryo Kawamura ◽  
Megan Littlehale ◽  
John F. Marko ◽  
Michael T. Laub
Keyword(s):  
High Resolution ◽  
Chromatin Immunoprecipitation ◽  
Binding Sites ◽  
Three Dimensional ◽  
Domain Model ◽  
Bacterial Protein ◽  
E Coli ◽  
Genome Wide ◽  
Powerful Approach ◽  
Positive Supercoiling

AbstractSupercoiling impacts DNA replication, transcription, protein binding to DNA, and the three-dimensional organization of chromosomes. However, there are currently no methods to directly interrogate or map positive supercoils, so their distribution in genomes remains unknown. Here, we describe a method, GapR-seq, based on the chromatin immunoprecipitation of GapR, a bacterial protein that preferentially recognizes overtwisted DNA, for generating high-resolution maps of positive supercoiling. Applying this method to E. coli and S. cerevisiae, we find that positive supercoiling is widespread, associated with transcription, and particularly enriched between convergently-oriented genes, consistent with the “twin-domain” model of supercoiling. In yeast, we also find positive supercoils associated with centromeres, cohesin binding sites, autonomously replicating sites, and the borders of R-loops (DNA-RNA hybrids). Our results suggest that GapR-seq is a powerful approach, likely applicable in any organism, to investigate aspects of chromosome structure and organization not accessible by Hi-C or other existing methods.

scholarly journals A unique chromatin complex occupies young α-satellite arrays of human centromeres

2015 ◽  
Vol 1 (1) ◽  
pp. e1400234 ◽  
Author(s):  
Jorja G. Henikoff ◽  
Jitendra Thakur ◽  
Sivakanthan Kasinathan ◽  
Steven Henikoff
Keyword(s):  
High Resolution ◽  
Tandem Repeat ◽  
Base Pair ◽  
Chromatin Immunoprecipitation ◽  
Sequence Data ◽  
Higher Order ◽  
Precise Positioning ◽  
Exact Sequences

The intractability of homogeneous α-satellite arrays has impeded understanding of human centromeres. Artificial centromeres are produced from higher-order repeats (HORs) present at centromere edges, although the exact sequences and chromatin conformations of centromere cores remain unknown. We use high-resolution chromatin immunoprecipitation (ChIP) of centromere components followed by clustering of sequence data as an unbiased approach to identify functional centromere sequences. We find that specific dimeric α-satellite units shared by multiple individuals dominate functional human centromeres. We identify two recently homogenized α-satellite dimers that are occupied by precisely positioned CENP-A (cenH3) nucleosomes with two ~100–base pair (bp) DNA wraps in tandem separated by a CENP-B/CENP-C–containing linker, whereas pericentromeric HORs show diffuse positioning. Precise positioning is largely maintained, whereas abundance decreases exponentially with divergence, which suggests that young α-satellite dimers with paired ~100-bp particles mediate evolution of functional human centromeres. Our unbiased strategy for identifying functional centromeric sequences should be generally applicable to tandem repeat arrays that dominate the centromeres of most eukaryotes.

scholarly journals High-resolution, genome-wide mapping of positive supercoiling in chromosomes

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Monica S Guo ◽  
Ryo Kawamura ◽  
Megan L Littlehale ◽  
John F Marko ◽  
Michael T Laub
Keyword(s):  
High Resolution ◽  
Chromatin Immunoprecipitation ◽  
Binding Sites ◽  
Three Dimensional ◽  
Domain Model ◽  
Bacterial Protein ◽  
E Coli ◽  
Genome Wide ◽  
Powerful Approach ◽  
Positive Supercoiling

Supercoiling impacts DNA replication, transcription, protein binding to DNA, and the three-dimensional organization of chromosomes. However, there are currently no methods to directly interrogate or map positive supercoils, so their distribution in genomes remains unknown. Here, we describe a method, GapR-seq, based on the chromatin immunoprecipitation of GapR, a bacterial protein that preferentially recognizes overtwisted DNA, for generating high-resolution maps of positive supercoiling. Applying this method to E. coli and S. cerevisiae, we find that positive supercoiling is widespread, associated with transcription, and particularly enriched between convergently-oriented genes, consistent with the 'twin-domain' model of supercoiling. In yeast, we also find positive supercoils associated with centromeres, cohesin binding sites, autonomously replicating sites, and the borders of R-loops (DNA-RNA hybrids). Our results suggest that GapR-seq is a powerful approach, likely applicable in any organism, to investigate aspects of chromosome structure and organization not accessible by Hi-C or other existing methods.

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