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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Feb 16, 2017 |
| Title |
Systematic investigation of transcription factor activity in the context of chromatin using massively parallel DNA binding and expression assays |
| Organism |
Saccharomyces cerevisiae |
| Experiment type |
Other
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| Summary |
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
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| Overall design |
A library of ~1600 promoter variants of length 210bp was designed and synthesized on Agilent programmable microarrays. Each sequence includes 19bp 5’ primer sequence, a 10bp unique barcode sequence, 163bp variable region and 18 bp 3’ primer. Sequences were integrated into the yeast genome upstream of a YFP reporter. Yeast population carrying this library was subjected to expression measurements as described in Sharon et al, Nature Biotechnology, 2012. In brief, cells were grown to mid-log and sorted in FACSAria to 16 bins based on YFP fluorescence. Sorted cells were grown to stationary phase, DNA was extracted and PCR amplification of the region of interest was performed. The 3′ primer was common to all bins (5′-NNNNNTTATGTGATAATGCCTAGGATCGC-3′, where the Ns represent random nucleotides). The 5′ primer had a common sequence and a unique upstream 5-bp barcode sequence (underlined) that was specific to each bin (5′-XXXXXGGGGACCAGGTGCCGTAAC-3′, where the Xs represent the bin's unique sequence). Three 5’ primers were used for the amplification of each bin, reads were later joined after we found them to be highly consistent. The list of primers is shown below. Amplified product was gel-extracted and 10 nanograms were used in library preparation for sequencing (protocol adopted from Blecher-Gonen et al. 2013). The DNA was amplified using 14 amplification cycles and library was analyzed using the 2200 TapeStation system and sequenced with 50-bp, single read flowcell on an Illumina HiSeq 2000 sequencer. This sample is referred as the “Expression measurements”. Yeast population carrying the library was subjected to a novel binding assay, aimed to reveal the occupancy pattern on the designed promoter variants. Cells were crosslinked and spheroplasts were then obtained. Spheroplasts were then subjected to methylation by the CpG Methyltranferase M.SssI. DNA was extracted and Bisulfite conversion was performed. We then PCR amplify the region of interest (520bp) with the following primers (that include 5bp tails underlined): 5’ primer – 5’-CATTCTAAAAGAATAGACCTAGATAGGGTT-3’, 3’ primer – 5’-NNNNNCATCACCATCTAATTCAACCAAAAT-3’. Amplified product was gel-extracted and 10 nanograms were used in library preparation for sequencing (protocol adopted from Blecher-Gonen et al. 2013). The DNA was amplified using 14 amplification cycles and library was analyzed using the 2200 TapeStation system. Libraries were sequenced on a 300 paired-end flow cells (MiSeq Reagent Kit v3, MS-102-3001) on MiSeq desktop sequencer (Illumina). This sample is referred as the “Binding measurements”. As the samples have low complexity they were mixed with other material prior to sequencing, the relevant reads can be identified by the sequence of the primers. Expression bins 5’ primers:
Bin1 AACGAGGGACCAGGTGCCGTAAC Bin2 GCATAGGGACCAGGTGCCGTAAC
Bin3 AGATAGGGACCAGGTGCCGTAAC Bin4 GAGTAGGGACCAGGTGCCGTAAC
Bin5 TCGTAGGGACCAGGTGCCGTAAC Bin6 CTGTAGGGACCAGGTGCCGTAAC
Bin7 TATTAGGGACCAGGTGCCGTAAC Bin8 CGTTAGGGACCAGGTGCCGTAAC
Bin9 ACGAAGGGACCAGGTGCCGTAAC Bin10 TAAGCGGGACCAGGTGCCGTAAC
Bin11 TTATCGGGACCAGGTGCCGTAAC Bin12 TACTCGGGACCAGGTGCCGTAAC
Bin13 CATTCGGGACCAGGTGCCGTAAC Bin14 CCTAAGGGACCAGGTGCCGTAAC
Bin15 GCAATGGGACCAGGTGCCGTAAC Bin16 CTACAGGGACCAGGTGCCGTAAC
Bin1 ACTATGGGACCAGGTGCCGTAAC Bin2 TACCAGGGACCAGGTGCCGTAAC
Bin3 CAAGTGGGACCAGGTGCCGTAAC Bin4 TGAGTGGGACCAGGTGCCGTAAC
Bin5 ATAGTGGGACCAGGTGCCGTAAC Bin6 AAGGTGGGACCAGGTGCCGTAAC
Bin7 ATCCAGGGACCAGGTGCCGTAAC Bin8 GATGTGGGACCAGGTGCCGTAAC
Bin9 TCTGTGGGACCAGGTGCCGTAAC Bin10 AGTGTGGGACCAGGTGCCGTAAC
Bin11 CTTGTGGGACCAGGTGCCGTAAC Bin12 TCATTGGGACCAGGTGCCGTAAC
Bin13 AAGCAGGGACCAGGTGCCGTAAC Bin14 CGATTGGGACCAGGTGCCGTAAC
Bin15 GTATTGGGACCAGGTGCCGTAAC Bin16 CACTTGGGACCAGGTGCCGTAAC
Bin1 ATGTTGGGACCAGGTGCCGTAAC Bin2 CATCAGGGACCAGGTGCCGTAAC
Bin3 TCTCAGGGACCAGGTGCCGTAAC Bin4 AGTCAGGGACCAGGTGCCGTAAC
Bin5 GTTCAGGGACCAGGTGCCGTAAC Bin6 ACAGAGGGACCAGGTGCCGTAAC
Bin7 GTAGAGGGACCAGGTGCCGTAAC Bin8 TTGGAGGGACCAGGTGCCGTAAC
Bin9 TGTGAGGGACCAGGTGCCGTAAC Bin10 ATTGAGGGACCAGGTGCCGTAAC
Bin11 GACAAGGGACCAGGTGCCGTAAC Bin12 CAATAGGGACCAGGTGCCGTAAC
Bin13 TCCAAGGGACCAGGTGCCGTAAC Bin14 AGCAAGGGACCAGGTGCCGTAAC
Bin15 CTCAAGGGACCAGGTGCCGTAAC Bin16 CAGAAGGGACCAGGTGCCGTAAC
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| Contributor(s) |
Levo M, Avnit-Sagi T, Lotan-Pompan M, Kalma Y, Weinberger A, Yakhini Z, Segal E |
| Citation(s) |
28212748 |
| Submission date |
Dec 12, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Michal Levo |
| Organization name |
Weizmann Institute of Science
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| Lab |
Eran Segal lab
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| Street address |
Herzl Street 234
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| City |
Rehovot, |
| ZIP/Postal code |
7610001 |
| Country |
Israel |
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| Platforms (2) |
| GPL13821 |
Illumina HiSeq 2000 (Saccharomyces cerevisiae) |
| GPL17143 |
Illumina MiSeq (Saccharomyces cerevisiae) |
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| Samples (2) |
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| Relations |
| BioProject |
PRJNA357133 |
| SRA |
SRP094981 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE92300_suppTable2.xlsx |
6.5 Mb |
(ftp)(http) |
XLSX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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