Dataset Information

Pull-down of nuclear proteins from HGC-27 cell line

ABSTRACT: To identify nuclear proteins that interact with KIT2KIT* G-quadruplex DNA, pull-down assays were performed with nuclear extracts from the KIT-positive HGC-27 cell line. Streptavidin-coated paramagnetic beads were derivatized with the biotinylated oligonucleotide and subsequently incubated with nuclear extracts. Bound proteins were eluted with a KCl gradient. A last fraction was obtained by boiling beads in denaturing Laemmli sample loading buffer. When solved by SDS-PAGE, this last fraction exhibited three main bands that were cut and subjected to in-gel trypsin digestion and LC-MSE analyses for protein identification. Two of them (bands S1 and S2 at ~15 kDa and ~28 kDa, respectively) corresponded to Streptavidin monomer and dimer that detached from the beads along boiling procedure. That aside, the band at ~50 kDa (band V) was associated to the intermediate filament protein Vimentin.

INSTRUMENT(S): Xevo G2-S Tof

ORGANISM(S): Homo Sapiens (human) Streptomyces Avidinii

TISSUE(S): Epithelial Cell

DISEASE(S): Stomach Carcinoma

SUBMITTER: Silvia Ceschi

LAB HEAD: Claudia Sissi

PROVIDER: PXD026505 | Pride | 2022-04-04

REPOSITORIES: Pride

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Vimentin binds to G-quadruplex repeats found at telomeres and gene promoters.

Ceschi Silvia S Berselli Michele M Cozzaglio Marta M Giantin Mery M Toppo Stefano S Spolaore Barbara B Sissi Claudia C

Nucleic acids research 20220201 3

G-quadruplex (G4) structures that can form at guanine-rich genomic sites, including telomeres and gene promoters, are actively involved in genome maintenance, replication, and transcription, through finely tuned interactions with protein networks. In the present study, we identified the intermediate filament protein Vimentin as a binder with nanomolar affinity for those G-rich sequences that give rise to at least two adjacent G4 units, named G4 repeats. This interaction is supported by the N-ter ...[more]

PMID: 35100428

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Project description:Binding of transcription factors (TFs) to regulatory sequences is a pivotal step in the control of gene expression. Despite many advances in the characterization of sequence motifs recognized by TFs, our ability to quantitatively predict TF binding to different regulatory sequences is still limited. Here, we present a novel experimental assay termed BunDLE-seq that provides quantitative measurements of TF binding to thousands of fully designed sequences of 200 bp in length within a single experiment. Applying this binding assay to two yeast TFs we demonstrate that sequences outside the core TF binding site profoundly affect TF binding. We show that TF-specific models based on the sequence or DNA shape of the regions flanking the core binding site are highly predictive of the measured differential TF binding. We further characterize the dependence of TF binding, accounting for measurements of single and co-occurring binding events, on the number and location of binding sites and on the TF concentration. Finally, by coupling our in vitro TF binding measurements, and another application of our method probing nucleosome formation, to in vivo expression measurements carried out with the same template sequences now serving and promoters, we offer insights into mechanisms that may determine the different expression outcomes observed. Our assay thus paves the way to a more comprehensive understanding of TF binding to regulatory sequences, and allows the characterization of TF binding determinants within and outside of core binding sites. Binding experiments were carried out with one of two input libraries; Lib1 (also referred to as “main lib”) includes designed sequence variants of length 150bp, and Lib2 (also referred to as “constant flanks lib”) includes designed sequence variants of length 200bp. The sequences of the variants of interest can be found in the second column of the processed data file (BunDLE-seq_data, see 3 excel sheets). EXP1, EXP2 and EXP3 were carried out using Lib1, and EXP4 was carried out using Lib2. Binding experiments were carried out with different proteins (in different concentrations) – DNA was extracted from bound bands that were formed on the gel and amplified with a primer with a unique 5bp upstream tail specifying the identity of the originating band (also referred to as “band barcode”). Data from the following bands was used in the analysis: EXP1_band1, no-protein band – with band barcode “AACGA”, EXP1_band2, single Gcn4 (conc 1:8) bound band* – with band barcode “TCGTA” , EXP1_band3, histones bound band – with band barcode “AGATA”, EXP2_band1, no-protein band – with band barcode “AACGA”, EXP2_band2, single Gcn4 (conc 1:8) bound band* – with band barcode “AGATA”, EXP2_band2, single Gcn4 (conc 1:7) bound band – with band barcode “CACTT”, EXP2_band2, single Gcn4 (conc 1:6) bound band – with band barcode “TCGTA”, EXP2_band2, single Gcn4 (conc 1:5) bound band – with band barcode “CATCA”, EXP2_band2, single Gcn4 (conc 1:4) bound band – with band barcode “TATTA”, EXP2_band2, single Gcn4 (conc 1:3) bound band – with band barcode “AGTCA”, EXP2_band2, single Gcn4 (conc 1:2) bound band – with band barcode “ACGAA”, EXP2_band2, single Gcn4 (conc 1:1) bound band – with band barcode “ACAGA”, EXP2_band2, two Gcn4 (conc 1:1) bound band – barcoded with “GTAGA”, EXP3_band1, no-protein band – with band barcode “GCATA”, EXP3_band2, single Gal4 (conc 1:1) bound band – with band barcode “GATGT”, EXP3_band3, single Gal4 (conc 3:1) bound band – with band barcode “GTTCA”, EXP3_band4, two Gal4 (conc 3:1) bound band – with band barcode “CTCAA”, EXP4_band1, no-protein band – with band barcode “GAGTA”, EXP4_band2, single Gcn4 (conc 1:3) bound band – “TACCA”, EXP4_band3, no-protein band – with band barcode “TCGTT”, EXP4_band3, single Gal4 (conc 3:1) bound band – with band barcode “ACATC”, *biological replicates

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			Action	DRS
	15n.mzid.gz	Mzid
	28n.mzid.gz	Mzid
	50n.mzid.gz	Mzid
	FTtoiGaSS.mgf	Mgf
	FTtoiGaSe.mgf	Mgf