PROBER Identifies Proteins Associated with Programable Sequence-Specific DNA in Living Cells
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive Plus
ORGANISM(S): Homo Sapiens (human)
SUBMITTER: 
Smarajit Mondal
LAB HEAD: Paul Khavari
PROVIDER: PXD029470 | Pride | 2022-07-27
REPOSITORIES: Pride
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Project description:Specific DNA-protein interactions mediate physiologic gene regulation and may be altered by DNA variants linked to polygenic disease. To enhance the speed and signal-to-noise ratio (SNR) of identifying and quantifying proteins associating with specific DNA sequences in living cells, we developed proximal biotinylation by episomal recruitment (PROBER). PROBER uses high copy episomes to amplify SNR along with proximity proteomics (BioID) to identify the transcription factors (TFs) and additional gene regulators associated with DNA sequences of interest. PROBER quantified steady-state and inducible association of TFs and associated chromatin regulators to target DNA sequences and quantified binding quantitative trait loci (bQTLs) due to single nucleotide variants. PROBER identified alterations in gene regulator associations due to cancer hotspot mutations in the hTERT promoter, indicating these mutations increase promoter association with specific gene activators. PROBER offers an approach to rapidly identify proteins associated with specific DNA sequences and their variants in living cells.
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Project description:DNA-protein interactions mediate physiologic gene regulation and may be altered by DNA variants linked to polygenic disease. To enhance the speed and signal-to-noise ratio (SNR) of identifying and quantifying proteins that associate with specific DNA sequences in living cells, we developed proximal biotinylation by episomal recruitment (PROBER). PROBER uses high copy episomes to amplify SNR along with proximity proteomics (BioID) to identify the transcription factors (TFs) and additional gene regulators associated with short DNA sequences of interest. PROBER quantified steady-state and inducible association of TFs and corresponding chromatin regulators to target DNA sequences as well as binding quantitative trait loci (bQTLs) due to single nucleotide variants. PROBER identified alterations in regulator associations due to cancer hotspot mutations in the hTERT promoter, indicating these mutations increase promoter association with specific gene activators. PROBER provides an approach to rapidly identify proteins associated with specific DNA sequences and their variants in living cells
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Project description:Abstract: Bacteria adapt to the constantly changing environments largely by transcriptional regulation through the activities of various transcription factors (TFs). However, techniques that monitor the in situ TF-promoter interactions in living bacteria are lacking. Herein, we developed a whole-cell TF-promoter binding assay based on the intermolecular Förster resonance energy transfer (FRET) between a fluorescent unnatural amino acid CouA which is genetically encoded into defined sites in TFs and the live cell fluorescent nucleic acid stain SYTO 9. We show that this new FRET pair monitors the intricate TF-promoter interactions elicited by various types of signal transduction systems with specificity and sensitivity. Furthermore, the assay is applicable to identify novel modulators of the regulatory systems of interest and monitor TF activities in bacteria colonized in C. elegans. In conclusion, we established a tractable and sensitive TF-promoter binding assay in living bacteria which not only complements currently available approaches for DNA-protein interactions but also provides novel opportunities for functional annotation of bacterial signal transduction systems and studies of the bacteria-host interface
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Project description:The DNA sequence preferences of the vast majority of eukaryotic transcription factors (TFs) are unknown. Using an approach designed to broadly sample both DNA-binding domain types and eukaryotic clades, we have determined DNA-binding motifs for 1,033 TFs from 131 diverse eukaryotes, encompassing 54 domain types. Closely related orthologs and paralogs typically have very similar sequence preferences; this property allows inference of motifs for roughly one third of the 166,851 known or predicted eukaryotic TFs. While the origins of most motifs can be dated to hundreds of millions of years ago, we also characterize more recent TF expansions. Sequences matching the motifs are enriched upstream of TSS in most eukaryotic lineages, and at informative eQTL SNPs in Arabidopsis promoters, demonstrating their utility in mapping transcriptional networks. The motifs are housed at http://cisbp.ccbr.utoronto.ca Protein binding microarray (PBM) experiments were performed for a set of 1048 diverse eukaryotic transcription factors. Briefly, the PBMs involved binding GST-tagged DNA-binding proteins to two double-stranded 44K Agilent microarrays, each containing a different DeBruijn sequence design, in order to determine their sequence preferences. Details of the PBM protocol are described in Berger et al., Nature Biotechnology 2006.
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