Unraveling determinants of transcription factor binding outside the core binding site
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ABSTRACT:
ORGANISM(S): synthetic construct
PROVIDER: GSE66143 | GEO | 2015/03/04
SECONDARY ACCESSION(S): PRJNA275965
REPOSITORIES: GEO
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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
2015-03-04 | E-GEOD-66143 | biostudies-arrayexpress
Project description:Precise gene expression patterns are established by timely and specific binding of transcription factors (TFs) to regulatory sequences. While these events occur in the context of chromatin, our understanding of how TF-nucleosome interplay affects gene expression is highly limited. Here we present a novel assay for high-resolution measurements of both DNA occupancy and gene expression on large-scale libraries of systematically designed regulatory sequences. Our assay is capable of revealing occupancy patterns at the single–cell level. It provides an accurate quantification of the fraction of the population bound by a nucleosome and captures distinct, even adjacent, TF binding events. By applying this assay to over 1500 promoter variants in yeast, we reveal pronounced differences in the dependency of TF activity on chromatin. Our approach provides means to classify TFs by their differential capacity to alter chromatin and promote expression. We further demonstrate how different regulatory sequences give rise to nucleosome-mediated TF collaborations that quantitatively account for the resulting expression. We show the utility of this approach in dissecting the logic of native regulatory sequences, highlighting the importance of TF-nucleosome interactions in the quantitative readout of regulatory information
2017-02-16 | GSE92300 | GEO
Project description:Transcription factors (TF) recognize specific genomic sequences to regulate complex gene expression programs. Although it is well established that TFs bind specific DNA sequences using a combination of base readout and shape recognition, some fundamental aspects of protein-DNA binding remain poorly understood. Many DNA-binding proteins induce changes in the DNA structure outside the intrinsic B-DNA envelope. However, how the energetic cost associated with distorting DNA contributes to recognition has proven difficult to study because the distorted DNA exists in low-abundance in the unbound ensemble. Here, we use a novel high-throughput assay called SaMBA (Saturation Mismatch-Binding Assay) to investigate the role of DNA conformational penalties in TF-DNA recognition. In SaMBA, mismatches are introduced to pre-induce DNA structural distortions much larger than those induced by changes in Watson-Crick sequence. Strikingly, approximately 10% of mismatches increased TF binding, and at least one mismatch was found that increased the binding affinity for each of 22 examined TFs. Mismatches also converted non-specific sites into high-affinity sites, and high-affinity sites into super-sites stronger than any known canonical binding site. Determination of high-resolution X-ray structures, combined with NMR measurements and structural analyses revealed that many of the mismatches that increase binding induce distortions similar to those induced by protein binding, thus pre-paying some of the energetic cost to deform the DNA. Our work indicates that conformational penalties are a major determinant of protein-DNA recognition, and reveals mechanisms by which mismatches can recruit TFs and thus modulate replication and repair activities in the cell.
2020-08-18 | GSE156373 | GEO
Project description:Transcription factors (TF) recognize specific genomic sequences to regulate complex gene expression programs. Although it is well established that TFs bind specific DNA sequences using a combination of base readout and shape recognition, some fundamental aspects of protein-DNA binding remain poorly understood. Many DNA-binding proteins induce changes in the DNA structure outside the intrinsic B-DNA envelope. However, how the energetic cost associated with distorting DNA contributes to recognition has proven difficult to study because the distorted DNA exists in low-abundance in the unbound ensemble. Here, we use a novel high-throughput assay called SaMBA (Saturation Mismatch-Binding Assay) to investigate the role of DNA conformational penalties in TF-DNA recognition. In SaMBA, mismatches are introduced to pre-induce DNA structural distortions much larger than those induced by changes in Watson-Crick sequence. Strikingly, approximately 10% of mismatches increased TF binding, and at least one mismatch was found that increased the binding affinity for each of 22 examined TFs. Mismatches also converted non-specific sites into high-affinity sites, and high-affinity sites into super-sites stronger than any known canonical binding site. Determination of high-resolution X-ray structures, combined with NMR measurements and structural analyses revealed that many of the mismatches that increase binding induce distortions similar to those induced by protein binding, thus pre-paying some of the energetic cost to deform the DNA. Our work indicates that conformational penalties are a major determinant of protein-DNA recognition, and reveals mechanisms by which mismatches can recruit TFs and thus modulate replication and repair activities in the cell.
2020-08-29 | GSE157065 | GEO
Project description:Transcription factors (TF) recognize specific genomic sequences to regulate complex gene expression programs. Although it is well established that TFs bind specific DNA sequences using a combination of base readout and shape recognition, some fundamental aspects of protein-DNA binding remain poorly understood. Many DNA-binding proteins induce changes in the DNA structure outside the intrinsic B-DNA envelope. However, how the energetic cost associated with distorting DNA contributes to recognition has proven difficult to study because the distorted DNA exists in low-abundance in the unbound ensemble. Here, we use a novel high-throughput assay called SaMBA (Saturation Mismatch-Binding Assay) to investigate the role of DNA conformational penalties in TF-DNA recognition. In SaMBA, mismatches are introduced to pre-induce DNA structural distortions much larger than those induced by changes in Watson-Crick sequence. Strikingly, approximately 10% of mismatches increased TF binding, and at least one mismatch was found that increased the binding affinity for each of 22 examined TFs. Mismatches also converted non-specific sites into high-affinity sites, and high-affinity sites into super-sites stronger than any known canonical binding site. Determination of high-resolution X-ray structures, combined with NMR measurements and structural analyses revealed that many of the mismatches that increase binding induce distortions similar to those induced by protein binding, thus pre-paying some of the energetic cost to deform the DNA. Our work indicates that conformational penalties are a major determinant of protein-DNA recognition, and reveals mechanisms by which mismatches can recruit TFs and thus modulate replication and repair activities in the cell.
2020-08-29 | GSE157064 | GEO
Project description:Transcription factors (TF) recognize specific genomic sequences to regulate complex gene expression programs. Although it is well established that TFs bind specific DNA sequences using a combination of base readout and shape recognition, some fundamental aspects of protein-DNA binding remain poorly understood. Many DNA-binding proteins induce changes in the DNA structure outside the intrinsic B-DNA envelope. However, how the energetic cost associated with distorting DNA contributes to recognition has proven difficult to study because the distorted DNA exists in low-abundance in the unbound ensemble. Here, we use a novel high-throughput assay called SaMBA (Saturation Mismatch-Binding Assay) to investigate the role of DNA conformational penalties in TF-DNA recognition. In SaMBA, mismatches are introduced to pre-induce DNA structural distortions much larger than those induced by changes in Watson-Crick sequence. Strikingly, approximately 10% of mismatches increased TF binding, and at least one mismatch was found that increased the binding affinity for each of 22 examined TFs. Mismatches also converted non-specific sites into high-affinity sites, and high-affinity sites into super-sites stronger than any known canonical binding site. Determination of high-resolution X-ray structures, combined with NMR measurements and structural analyses revealed that many of the mismatches that increase binding induce distortions similar to those induced by protein binding, thus pre-paying some of the energetic cost to deform the DNA. Our work indicates that conformational penalties are a major determinant of protein-DNA recognition, and reveals mechanisms by which mismatches can recruit TFs and thus modulate replication and repair activities in the cell.
2020-08-16 | GSE156304 | GEO
Project description:Transcription factors (TF) recognize specific genomic sequences to regulate complex gene expression programs. Although it is well established that TFs bind specific DNA sequences using a combination of base readout and shape recognition, some fundamental aspects of protein-DNA binding remain poorly understood. Many DNA-binding proteins induce changes in the DNA structure outside the intrinsic B-DNA envelope. However, how the energetic cost associated with distorting DNA contributes to recognition has proven difficult to study because the distorted DNA exists in low-abundance in the unbound ensemble. Here, we use a novel high-throughput assay called SaMBA (Saturation Mismatch-Binding Assay) to investigate the role of DNA conformational penalties in TF-DNA recognition. In SaMBA, mismatches are introduced to pre-induce DNA structural distortions much larger than those induced by changes in Watson-Crick sequence. Strikingly, approximately 10% of mismatches increased TF binding, and at least one mismatch was found that increased the binding affinity for each of 22 examined TFs. Mismatches also converted non-specific sites into high-affinity sites, and high-affinity sites into super-sites stronger than any known canonical binding site. Determination of high-resolution X-ray structures, combined with NMR measurements and structural analyses revealed that many of the mismatches that increase binding induce distortions similar to those induced by protein binding, thus pre-paying some of the energetic cost to deform the DNA. Our work indicates that conformational penalties are a major determinant of protein-DNA recognition, and reveals mechanisms by which mismatches can recruit TFs and thus modulate replication and repair activities in the cell.
2020-08-29 | GSE157067 | GEO
Project description:Transcription factors (TF) recognize specific genomic sequences to regulate complex gene expression programs. Although it is well established that TFs bind specific DNA sequences using a combination of base readout and shape recognition, some fundamental aspects of protein-DNA binding remain poorly understood. Many DNA-binding proteins induce changes in the DNA structure outside the intrinsic B-DNA envelope. However, how the energetic cost associated with distorting DNA contributes to recognition has proven difficult to study because the distorted DNA exists in low-abundance in the unbound ensemble. Here, we use a novel high-throughput assay called SaMBA (Saturation Mismatch-Binding Assay) to investigate the role of DNA conformational penalties in TF-DNA recognition. In SaMBA, mismatches are introduced to pre-induce DNA structural distortions much larger than those induced by changes in Watson-Crick sequence. Strikingly, approximately 10% of mismatches increased TF binding, and at least one mismatch was found that increased the binding affinity for each of 22 examined TFs. Mismatches also converted non-specific sites into high-affinity sites, and high-affinity sites into super-sites stronger than any known canonical binding site. Determination of high-resolution X-ray structures, combined with NMR measurements and structural analyses revealed that many of the mismatches that increase binding induce distortions similar to those induced by protein binding, thus pre-paying some of the energetic cost to deform the DNA. Our work indicates that conformational penalties are a major determinant of protein-DNA recognition, and reveals mechanisms by which mismatches can recruit TFs and thus modulate replication and repair activities in the cell.
2020-08-29 | GSE157066 | GEO
Project description:DNA sequence is a major determinant of the binding specificity of transcription factors (TFs) for their genomic targets. However, eukaryotic cells often express, at the same time, TFs with highly similar DNA binding motifs but distinct in vivo targets. Currently, it is not well understood how TFs with seemingly identical DNA motifs achieve unique specificities in vivo. Here, we used custom protein binding microarrays to analyze TF specificity for putative binding sites in their genomic sequence context. Using yeast TFs Cbf1 and Tye7 as our case study, we found that binding sites of these bHLH TFs (i.e., E-boxes) are bound differently in vitro and in vivo, depending on their genomic context. Computational analyses suggest that nucleotides outside E-box binding sites contribute to specificity by influencing the 3D structure of DNA binding sites. Thus, local shape of target sites might play a widespread role in achieving regulatory specificity within TF families. Two protein binding microarray (PBM) experiments of Saccharomyces cerevisiae transcription factors were performed. Briefly, the PBMs involved binding GST-tagged yeast transcription factors Cbf1 and Tye7 to double-stranded 44K Agilent microarrays in order to determine the accuracy of our regression models for TF-DNA binding specificity. Briefly, this array contains 30-bp genomic sequences from our initial custom array (Gordan et al 2013, submitted), with 0 through 4 mutations designed at various positions in the genomic sequences. Each sequence in represented in 6 replicate spots. We report the PBM signal intensity for each spot. The PBM protocol is described in Berger et al., Nature Biotechnology 2006 (PMID 16998473).
2013-03-29 | E-GEOD-44437 | biostudies-arrayexpress
Project description:Despite much research, our understanding of the architecture and cis-regulatory elements of human promoters is still lacking. Here, we devised a high-throughput assay to quantify the activity of ~15,000 fully designed sequences that we integrated and expressed from a fixed location within the human genome. We used this method to investigate thousands of native promoters and pre-initiation complex (PIC) binding regions followed by in-depth characterization of the sequence motifs underlying promoter activity including core promoter elements and TF binding-sites. We find that core promoters drive transcription mostly unidirectionally, and that sequences originating from promoters exhibit stronger activity than those originating from enhancers. Testing multiple synthetic configurations of core promoter elements, we dissect the motifs that positively and negatively regulate transcription as well as the effect of their combinations and distances, including a 10bp periodicity in the optimal distance between the TATA and the Initiator. By comprehensively screening 133 TF binding-sites, we find that in contrast to core promoters, TF binding-sites maintain similar activity levels in both orientations supporting a model by which divergent transcription is driven by two distinct unidirectional core promoters sharing bidirectional TF binding-sites. Finally, we find a striking agreement between the effect of binding site multiplicity of individual TFs in our assay and their tendency to appear in homotypic clusters throughout the genome. Overall, our study systematically assays the elements that drive expression in core- and proximal- promoter regions and shed light on organization principles of regulatory regions in the human genome.
2018-08-08 | GSE118242 | GEO