A ChIP-exo screen of 887 Protein Capture Reagents Program transcription factor antibodies in human cells

Antibodies offer a powerful means to interrogate specific proteins in a complex milieu. However, antibody availability and reliability can be problematic, whereas epitope tagging can be impractical in many cases. To address these limitations, the Protein Capture Reagents Program (PCRP) generated over a thousand renewable monoclonal antibodies (mAbs) against human presumptive chromatin proteins. However, these reagents have not been widely field-tested. We therefore performed a screen to test their ability to enrich genomic regions via chromatin immunoprecipitation (ChIP) and a variety of orthogonal assays. Eight hundred eighty-seven unique antibodies against 681 unique human transcription factors (TFs) were assayed by ultra-high-resolution ChIP-exo/seq, generating approximately 1200 ChIP-exo data sets, primarily in a single pass in one cell type (K562). Subsets of PCRP mAbs were further tested in ChIP-seq, CUT&RUN, STORM super-resolution microscopy, immunoblots, and protein binding microarray (PBM) experiments. About 5% of the tested antibodies displayed high-confidence target (i.e., cognate antigen) enrichment across at least one assay and are strong candidates for additional validation. An additional 34% produced ChIP-exo data that were distinct from background and thus warrant further testing. The remaining 61% were not substantially different from background, and likely require consideration of a much broader survey of cell types and/or assay optimizations. We show and discuss the metrics and challenges to antibody validation in chromatin-based assays.

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Screening of PCRP transcription factor antibodies in biochemical assays

10.1101/2020.06.08.140046 ◽

2020 ◽

Author(s):

William K. M. Lai ◽

Luca Mariani ◽

Gerson Rothschild ◽

Edwin R. Smith ◽

Bryan J. Venters ◽

...

Keyword(s):

Super Resolution ◽

Protein Capture ◽

Protein Binding Microarray ◽

Biochemical Assays ◽

Powerful Means ◽

Specific Proteins ◽

Chromatin Proteins ◽

Super Resolution Microscopy ◽

Antibody Validation ◽

Improve Reliability

AbstractAntibodies offer a powerful means to interrogate specific proteins in a complex milieu, and where epitope tagging is impractical. However, antibody availability and reliability are problematic. The Protein Capture Reagents Program (PCRP) generated over a thousand renewable monoclonal antibodies against human-presumptive chromatin proteins in an effort to improve reliability. However, these reagents have not been widely field-tested. We therefore screened their ability in a variety of assays. 887 unique antibodies against 681 unique chromatin proteins, of which 605 are putative sequence-specific transcription factors (TFs), were assayed by ChIP-exo. Subsets were further tested in ChIP-seq, CUT&RUN, STORM super-resolution microscopy, immunoblots, and protein binding microarray (PBM) experiments. At least 6% of the tested antibodies were validated for use in ChIP-based assays by the most stringent of our criteria. An additional 34% produced data suggesting they warranted further testing for clearer validation. We demonstrate and discuss the metrics and limitations to antibody validation in chromatin-based assays.

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Single-Molecule Super-Resolution Microscopy Reveals Heteromeric Complexes of MET and EGFR upon Ligand Activation

International Journal of Molecular Sciences ◽

10.3390/ijms21082803 ◽

2020 ◽

Vol 21 (8) ◽

pp. 2803 ◽

Cited By ~ 4

Author(s):

Marie-Lena I.E. Harwardt ◽

Mark S. Schröder ◽

Yunqing Li ◽

Sebastian Malkusch ◽

Petra Freund ◽

...

Keyword(s):

Single Molecule ◽

Tyrosine Kinases ◽

Growth Factor Receptor ◽

Super Resolution ◽

Cell Types ◽

Surface Expression ◽

Living Cells ◽

Ligand Activation ◽

Receptor Clusters ◽

Super Resolution Microscopy

Receptor tyrosine kinases (RTKs) orchestrate cell motility and differentiation. Deregulated RTKs may promote cancer and are prime targets for specific inhibitors. Increasing evidence indicates that resistance to inhibitor treatment involves receptor cross-interactions circumventing inhibition of one RTK by activating alternative signaling pathways. Here, we used single-molecule super-resolution microscopy to simultaneously visualize single MET and epidermal growth factor receptor (EGFR) clusters in two cancer cell lines, HeLa and BT-20, in fixed and living cells. We found heteromeric receptor clusters of EGFR and MET in both cell types, promoted by ligand activation. Single-protein tracking experiments in living cells revealed that both MET and EGFR respond to their cognate as well as non-cognate ligands by slower diffusion. In summary, for the first time, we present static as well as dynamic evidence of the presence of heteromeric clusters of MET and EGFR on the cell membrane that correlates with the relative surface expression levels of the two receptors.

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A map of direct TF–DNA interactions in the human genome

Nucleic Acids Research ◽

10.1093/nar/gky1210 ◽

2018 ◽

Vol 47 (4) ◽

pp. e21-e21 ◽

Cited By ~ 19

Author(s):

Marius Gheorghe ◽

Geir Kjetil Sandve ◽

Aziz Khan ◽

Jeanne Chèneby ◽

Benoit Ballester ◽

...

Keyword(s):

Human Genome ◽

Protein Interaction Data ◽

Data Sets ◽

Dna Interactions ◽

Regulatory Modules ◽

Protein Protein Interaction ◽

Protein Binding Microarray ◽

Bona Fide ◽

Genomic Regions

Abstract Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is the most popular assay to identify genomic regions, called ChIP-seq peaks, that are bound in vivo by transcription factors (TFs). These regions are derived from direct TF–DNA interactions, indirect binding of the TF to the DNA (through a co-binding partner), nonspecific binding to the DNA, and noise/bias/artifacts. Delineating the bona fide direct TF–DNA interactions within the ChIP-seq peaks remains challenging. We developed a dedicated software, ChIP-eat, that combines computational TF binding models and ChIP-seq peaks to automatically predict direct TF–DNA interactions. Our work culminated with predicted interactions covering >2% of the human genome, obtained by uniformly processing 1983 ChIP-seq peak data sets from the ReMap database for 232 unique TFs. The predictions were a posteriori assessed using protein binding microarray and ChIP-exo data, and were predominantly found in high quality ChIP-seq peaks. The set of predicted direct TF–DNA interactions suggested that high-occupancy target regions are likely not derived from direct binding of the TFs to the DNA. Our predictions derived co-binding TFs supported by protein-protein interaction data and defined cis-regulatory modules enriched for disease- and trait-associated SNPs. We provide this collection of direct TF–DNA interactions and cis-regulatory modules through the UniBind web-interface (http://unibind.uio.no).

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CAGEd-oPOSSUM: motif enrichment analysis from CAGE-derived TSSs

10.1101/040667 ◽

2016 ◽

Cited By ~ 1

Author(s):

David J. Arenillas ◽

Alistair R.R. Forrest ◽

Hideya Kawaji ◽

Timo Lassman ◽

Wyeth W. Wasserman ◽

...

Keyword(s):

Large Scale ◽

Enrichment Analysis ◽

Cell Types ◽

Specific Cell ◽

Data Sets ◽

Transcription Start Sites ◽

Supplementary Material ◽

Supplementary Text ◽

Cap Analysis ◽

Genomic Regions

AbstractSummaryWith the emergence of large-scale Cap Analysis of Gene Expression (CAGE) data sets from individual labs and the FANTOM consortium, one can now analyze the cis-regulatory regions associated with gene transcription at an unprecedented level of refinement. By coupling transcription factor binding site (TFBS) enrichment analysis with CAGE-derived genomic regions, CAGEd-oPOSSUM can identify TFs that act as key regulators of genes involved in specific mammalian cell and tissue types. The webtool allows for the analysis of CAGE-derived transcription start sites (TSSs) either provided by the user or selected from ~1,300 mammalian samples from the FANTOM5 project with pre-computed TFBS predicted with JASPAR TF binding profiles. The tool helps power insights into the regulation of genes through the study of the specific usage of TSSs within specific cell types and/or under specific conditions.Availability and implementationThe CAGEd-oPOSUM web tool is implemented in Perl, MySQL, and Apache and is available at http://cagedop.cmmt.ubc.ca/CAGEd_oPOSSUM.Supporting InformationSupplementary Text, Figures, and Data are available online at bioRxiv.

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Self-labeling of proteins with chemical fluorescent dyes in BY-2 cells and Arabidopsis seedlings

10.1101/2020.03.09.983924 ◽

2020 ◽

Author(s):

Ryu J. Iwatate ◽

Akira Yoshinari ◽

Noriyoshi Yagi ◽

Marek Grzybowski ◽

Hiroaki Ogasawara ◽

...

Keyword(s):

Fluorescent Dyes ◽

Super Resolution ◽

Cell Plate ◽

Protein Labeling ◽

Synthetic Dyes ◽

Biological Processes ◽

Major Drawback ◽

Specific Proteins ◽

Super Resolution Microscopy

AbstractSynthetic chemical fluorescent dyes are promising tools for many applications in biology. SNAP tagging provides a unique opportunity for labeling of specific proteins in vivo with synthetic dyes for studying for example endocytosis, or super-resolution microscopy. However, despite the potential, chemical dye tagging has not been used effectively in plants. A major drawback was the limited knowledge regarding cell wall and membrane permeability of synthetic dyes. Twenty-six out of 31 synthetic dyes were taken up into BY-2 cells, eight were not taken up and can thus serve for measuring endocytosis. Three of the dyes that were able to enter the cells, SNAP-tag ligands of diethylaminocoumarin, tetramethylrhodamine (TMR) and silicon-rhodamine (SiR) 647 were used to SNAP tag α-tubulin. Successful tagging was verified by live cell imaging and visualization of microtubules arrays in interphase and during mitosis. Fluorescence activation-coupled protein labeling (FAPL) with DRBG-488 was used to observe PIN2 endocytosis and delivery to the vacuole as well as preferential delivery of newly synthesized PIN2 to the newly forming cell plate during mitosis. Together the data demonstrate that specific self-labeling of proteins can be used effectively in plants to study a wide variety to cell biological processes.

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The endoplasmic reticulum adopts two distinct forms of tubules

10.21203/rs.3.rs-753780/v1 ◽

2021 ◽

Author(s):

Ke Xu ◽

Bowen Wang ◽

Zhiheng Zhao ◽

Michael Xiong ◽

Rui Yan

Keyword(s):

Endoplasmic Reticulum ◽

Lipid Synthesis ◽

Super Resolution ◽

Cell Types ◽

Membrane Curvature ◽

Calcium Storage ◽

Complex Membrane ◽

Functional Implications ◽

Different Cell Types ◽

Super Resolution Microscopy

Abstract Being the largest and most expansive organelle in the cell, the endoplasmic reticulum (ER) carries diverse key functions from protein and lipid synthesis, protein folding and modification, transport, calcium storage, to organelle interactions1-6. The shaping mechanism of this complex, membrane-bounded organelle is thus of fundamental significance7-21. Using super-resolution microscopy, we uncover the coexistence of two distinct, well-defined forms of ER tubules in the mammalian cell. Whereas an ultrathin form, R1, is consistently covered by the membrane curvature-promoting protein Rtn4, in the second form, R2, Rtn4 curiously appears as two parallel lines at a conserved separation of ~105 nm over long ranges. The two tubule forms together account for ~90% of the total tubule lengths, with either one being dominant in different cell types. The R1-R2 dichotomy and the final tubule geometry are both co-regulated by Rtn4 and the ER sheet-maintaining protein Climp63, which respectively define the edge curvature and lumen height of the R2 tubules to generate a ribbon-like structure of well-defined width. The R1 and R2 tubules undergo active remodeling in the cell as they differently accommodate proteins, with the former effectively excluding ER-luminal proteins and ER-membrane proteins with large intraluminal domains. We thus unveil a dynamic ER-tubule structural dichotomy with intriguing functional implications.

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The endoplasmic reticulum adopts two distinct tubule forms

10.1101/2021.10.26.465969 ◽

2021 ◽

Author(s):

Bowen Wang ◽

Zhiheng Zhao ◽

Michael Xiong ◽

Rui Yan ◽

Ke Xu

Keyword(s):

Endoplasmic Reticulum ◽

Super Resolution ◽

Cell Types ◽

Membrane Curvature ◽

Parallel Lines ◽

Native Cell ◽

Functional Implications ◽

Different Cell Types ◽

Super Resolution Microscopy ◽

Er Membrane

The endoplasmic reticulum (ER) is a versatile organelle with diverse functions. Through super-resolution microscopy, we show that the peripheral ER in the mammalian cell adopts two distinct forms of tubules. Whereas an ultrathin form, R1, is consistently covered by ER-membrane curvature-promoting proteins, e.g., Rtn4 in the native cell, in the second form, R2, Rtn4 and analogs are arranged into two parallel lines at a conserved separation of ~105 nm over long ranges. The two tubule forms together account for ~90% of the total tubule length in the cell, with either one being dominant in different cell types. The R1-R2 dichotomy and the final tubule geometry are both co-regulated by Rtn4 (and analogs) and the ER sheet-maintaining protein Climp63, which respectively define the edge curvature and lumen height of the R2 tubules to generate a ribbon-like structure of well-defined width. Accordingly, the R2 tubule width correlates positively with the Climp63 intralumenal size. The R1 and R2 tubules undergo active remodeling at the second/sub-second time scales as they differently accommodate proteins, with the former effectively excluding ER-luminal proteins and ER-membrane proteins with large intraluminal domains. We thus uncover a dynamic structural dichotomy for ER tubules with intriguing functional implications.

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Quantitative super-resolution single molecule microscopy dataset of YFP-tagged growth factor receptors

10.1101/246488 ◽

2018 ◽

Author(s):

Tomáš Lukeš ◽

Jakub Pospíšil ◽

Karel Fliegel ◽

Theo Lasser ◽

Guy M. Hagen

Keyword(s):

Data Analysis ◽

Growth Factor ◽

Single Molecule ◽

Super Resolution ◽

Growth Factor Receptors ◽

Data Sets ◽

Localization Microscopy ◽

Super Resolution Microscopy ◽

Resolution Single ◽

Single Molecule Localization Microscopy

BackgroundSuper-resolution single molecule localization microscopy (SMLM) is a method for achieving resolution beyond the classical limit in optical microscopes (approx. 200 nm laterally). Yellow fluorescent protein (YFP) has been used for super-resolution single molecule localization microscopy, but less frequently than other fluorescent probes. Working with YFP in SMLM is a challenge because a lower number of photons are emitted per molecule compared to organic dyes which are more commonly used. Publically available experimental data can facilitate development of new data analysis algorithms.FindingsFour complete, freely available single molecule super-resolution microscopy datasets on YFP-tagged growth factor receptors expressed in a human cell line are presented including both raw and analyzed data. We report methods for sample preparation, for data acquisition, and for data analysis, as well as examples of the acquired images. We also analyzed the SMLM data sets using a different method: super-resolution optical fluctuation imaging (SOFI). The two modes of analysis offer complementary information about the sample. A fifth single molecule super-resolution microscopy dataset acquired with the dye Alexa 532 is included for comparison purposes.ConclusionThis dataset has potential for extensive reuse. Complete raw data from SMLM experiments has typically not been published. The YFP data exhibits low signal to noise ratios, making data analysis a challenge. These data sets will be useful to investigators developing their own algorithms for SMLM, SOFI, and related methods. The data will also be useful for researchers investigating growth factor receptors such as ErbB3.

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A map of direct TF-DNA interactions in the human genome

10.1101/394205 ◽

2018 ◽

Cited By ~ 2

Author(s):

Marius Gheorghe ◽

Geir Kjetil Sandve ◽

Aziz Khan ◽

Jeanne Chèneby ◽

Benoit Ballester ◽

...

Keyword(s):

Human Genome ◽

Protein Interaction Data ◽

Data Sets ◽

Dna Interactions ◽

Regulatory Modules ◽

Protein Protein Interaction ◽

Protein Binding Microarray ◽

Bona Fide ◽

Genomic Regions

ABSTRACTChromatin immunoprecipitation followed by sequencing (ChIP-seq) is the most popular assay to identify genomic regions, called ChIP-seq peaks, that are bound in vivo by transcription factors (TFs). These regions are derived from direct TF-DNA interactions, indirect binding of the TF to the DNA (through a co-binding partner), nonspecific binding to the DNA, and noise/bias/artifacts. Delineating the bona fide direct TF-DNA interactions within the ChIP-seq peaks remains challenging. We developed a dedicated software, ChIP-eat, that combines computational TF binding models and ChIP-seq peaks to automatically predict direct TF-DNA interactions. Our work culminated with predicted interactions covering >4% of the human genome, obtained by uniformly processing 1,983 ChIP-seq peak data sets from the ReMap database for 232 unique TFs. The predictions were a posteriori assessed using protein binding microarray and ChIP-exo data, and were predominantly found in high quality ChIP-seq peaks. The set of predicted direct TF-DNA interactions suggested that high-occupancy target regions are likely not derived from direct binding of the TFs to the DNA. Our predictions derived co-binding TFs supported by protein-protein interaction data and defined cis-regulatory modules enriched for disease- and trait-associated SNPs. Finally, we provide this collection of direct TF-DNA interactions and cis-regulatory modules in the human genome through the UniBind web-interface (http://unibind.uio.no).

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Synergy between B cell receptor/antigen uptake and MHCII peptide editing relies on HLA-DO tuning

Scientific Reports ◽

10.1038/s41598-019-50455-y ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 2

Author(s):

Wei Jiang ◽

Lital N. Adler ◽

Henriette Macmillan ◽

Elizabeth D. Mellins

Keyword(s):

B Cells ◽

B Cell ◽

Super Resolution ◽

Cell Types ◽

Cell Receptor ◽

Antigen Uptake ◽

Critical Pathways ◽

High Affinity ◽

Model Cell ◽

Super Resolution Microscopy

Abstract B cell receptors and surface-displayed peptide/MHCII complexes constitute two key components of the B-cell machinery to sense signals and communicate with other cell types during antigen-triggered activation. However, critical pathways synergizing antigen-BCR interaction and antigenic peptide-MHCII presentation remain elusive. Here, we report the discovery of factors involved in establishing such synergy. We applied a single-cell measure coupled with super-resolution microscopy to investigate the integrated function of two lysosomal regulators for peptide loading, HLA-DM and HLA-DO. In model cell lines and human tonsillar B cells, we found that tunable DM/DO stoichiometry governs DMfree activity for exchange of placeholder CLIP peptides with high affinity MHCII ligands. Compared to their naïve counterparts, memory B cells with less DMfree concentrate a higher proportion of CLIP/MHCII in lysosomal compartments. Upon activation mediated by high affinity BCR, DO tuning is synchronized with antigen internalization and rapidly potentiates DMfree activity to optimize antigen presentation for T-cell recruitment.

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