Hitchhiker antigens: Inconsistent ChIP results, questionable immunohistology data, and poor antibody performance may have a common factor

Parseghian, Missag H.

doi:10.1139/bcb-2013-0059

Cited by 13 publications

(8 citation statements)

References 57 publications

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“…Our results, yet again [29,55,58,59], emphasise the need for critical examination and extensive testing of antibodies prior to their experimental usage. Whenever possible, controls in ChIP-seq experiments should be performed by ChIP-ing of protein in a system where the protein is not physically present, as implemented in Knockout Implemented Normalization method (KOIN) [60].…”

Section: Discussionmentioning

confidence: 59%

HOT or not: examining the basis of high-occupancy target regions

Wreczycka

Franke

Uyar

et al. 2017

Preprint

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High-occupancy target (HOT) regions are the segments of the genome with unusually high number of transcription factor binding sites. These regions are observed in multiple species and thought to have biological importance due to high transcription factor occupancy. Furthermore, they coincide with house-keeping gene promoters and the associated genes are stably expressed across multiple cell types. Despite these features, HOT regions are solemnly defined using ChIP-seq experiments and shown to lack canonical motifs for transcription factors that are thought to be bound there. Although, ChIP-seq experiments are the golden standard for finding genome-wide binding sites of a protein, they are not noise free. Here, we show that HOT regions are likely to be ChIP-seq artifacts and they are similar to previously proposed 'hyper-ChIPable' regions. Using ChIP-seq data sets for knocked-out transcription factors, we demonstrate presence of false positive signals on HOT regions. We observe sequence characteristics and genomic features that are discriminatory of HOT regions, such as GC/CpGrich k-mers and enrichment of RNA-DNA hybrids (R-loops) and DNA tertiary structures (Gquadruplex DNA). The artificial ChIP-seq enrichment on HOT regions could be associated to these discriminatory features. Furthermore, we propose strategies to deal with such artifacts for the future ChIP-seq studies.

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Section: Discussionmentioning

confidence: 59%

HOT or not: examining the basis of high-occupancy target regions

Wreczycka

Franke

Uyar

et al. 2017

Preprint

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“…and the methods used for data analysis (e.g., peak calling). Third, ChIP-seq data contain numerous technical biases (Kidder et al, 2011), including formaldehyde crosslinking bias (Solomon and Varshavsky, 1985; Lu et al, 2010; Gavrilov et al, 2015), antibody specificity and variability problems (Parseghian, 2013; Schonbrunn, 2014; Wardle and Tan, 2015) (Figure S15), technical artifacts due to highly expressed regions of the genome (which are not corrected by regular input controls) (Teytelman et al, 2013; Park et al, 2013; Jain et al, 2015), bias due to genome fragmentation and PCR amplification (Bardet et al, 2011; Poptsova et al, 2014), etc. These biases can lead to false-positive and false-negative peaks, and they also significantly affect any quantitative estimates of in vivo TF binding levels derived from ChIP-seq data, in ways that we do not understand well enough to correct (Gavrilov et al, 2015).…”

Section: Resultsmentioning

confidence: 99%

Divergence in DNA Specificity among Paralogous Transcription Factors Contributes to Their Differential In Vivo Binding

et al. 2018

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SUMMARY Paralogous transcription factors (TFs) are oftentimes reported to have identical DNA-binding motifs, despite the fact that they perform distinct regulatory functions. Differential genomic targeting by paralogous TFs is generally assumed to be due to interactions with protein co-factors or the chromatin environment. Using a computational-experimental framework called iMADS (integrative modeling and analysis of differential specificity), we show that, contrary to previous assumptions, paralogous TFs bind differently to genomic target sites even in vitro. We used iMADS to quantify, model, and analyze specificity differences between 11 TFs from 4 protein families. We found that paralogous TFs have diverged mainly at mediumand low-affinity sites, which are poorly captured by current motif models. We identify sequence and shape features differentially preferred by paralogous TFs, and we show that the intrinsic differences in specificity among paralogous TFs contribute to their differential in vivo binding. Thus, our study represents a step forward in deciphering the molecular mechanisms of differential specificity in TF families.

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“…It is known that the total IgG purified on PtG sorbents is enriched by immune complexes and contains a lot of so-called “hitchhiker” ligands [42, 43]. We believe that this phenomenon is akin to the HAbs formation or is closely related to it, and many hitchhikers unidentified and untested as yet may be present in HAbs, including TF-positive ligands of tumor origin such as MUC1 which is overexpressed and aberrantly glycosylated in cancer, and identified as an antigenic component in IgG immune complexes in cancer patients [44, 45].…”

Section: Discussionmentioning

confidence: 99%

Hidden IgG Antibodies to the Tumor-Associated Thomsen-Friedenreich Antigen in Gastric Cancer Patients: Lectin Reactivity, Avidity, and Clinical Relevance

Kurtenkov

Klaamas

2017

BioMed Research International

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Natural antibodies to the tumor-associated Thomsen-Friedenreich antigen (TF) are related to tumor immunosurveillance and cancer patients' survival. Hidden IgG antibodies (HAbs) to TF, their lectin reactivity, avidity, and clinical relevance were studied. HAbs were present in cancer patients and controls. A decreased level of IgG HAbs was detected in cancer. The HAbs level positively correlated with the sialospecific SNA lectin binding in purified total IgG (tIgG) in donors and cancer patients, indicating that HAbs are higher sialylated. The avidity of anti-TF IgG in tIgG samples was lower in cancer patients (P = 0.025) while no difference in the avidity of free anti-TF IgG was established. A negative correlation between the avidity of anti-TF IgG in tIgG and SNA binding in both groups was observed (P < 0.0001). The HAbs level negatively correlated with the anti-TF IgG avidity in tIgG only in donors (P = 0.003). Changes in the level of HAbs and Abs avidity showed a rather good stage- and gender-dependent diagnostic accuracy. Cancer patients with a lower anti-TF IgG avidity in tIgG showed a benefit in survival. Thus the TF-specific HAbs represent a particular subset of anti-TF IgG that differ from free serum anti-TF IgG in SNA reactivity, avidity, diagnostic potential, and relation to survival.

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Hitchhiker antigens: Inconsistent ChIP results, questionable immunohistology data, and poor antibody performance may have a common factor

Cited by 13 publications

References 57 publications

HOT or not: examining the basis of high-occupancy target regions

HOT or not: examining the basis of high-occupancy target regions

Divergence in DNA Specificity among Paralogous Transcription Factors Contributes to Their Differential In Vivo Binding

Hidden IgG Antibodies to the Tumor-Associated Thomsen-Friedenreich Antigen in Gastric Cancer Patients: Lectin Reactivity, Avidity, and Clinical Relevance

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