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Series GSE159989 Query DataSets for GSE159989
Status Public on Jul 02, 2022
Title DNA binding specificity for the bHLH-PAS transcription factor family
Organism synthetic construct
Experiment type Other
Summary Transcription factor-DNA interactions and their specificities have been described for many different classes of transcription factor families. However, heterodimeric transcription factor complexes still remain poorly characterised. The basic-Helix-Loop-Helix (bHLH) transcription factor family is one of the largest transcription factor families that typically bind DNA though a degenerate CANNTG elements as heterodimers or homodimers. Here we characterise the DNA binding of the bHLH - Per-Arnt-Sim (PAS) (bHLH-PAS) domain containing transcription factor family using SELEX-high-throughput sequencing coupled with quantitative computational modelling analysis. We show that most dimeric bHLH-PAS transcription factors bind to distinct core NNCGTG response elements but bind over a much larger footprint than previously characterised. Modelled DNA-protein interactions were found to correlate with structural analysis, DNA shape predictions and in vivo transcription factor occupancy.
 
Overall design We determined the DNA binding specificity of the bHLH-PAS transcription factors by SELEX-seq and No-Read-Left Behind (NRLB) Protein-DNA Modelling. We purified full length human ARNT (1-774) or ARNT2 (1-717) homodimers from Expi293 cells, bHLH-PAS (PAS A and PAS B, N-terminus) heterdimers (human HIF1a(1-375)/ARNT(1-474), human HIF2a(1-353)/ARNT(1-474), human SIM1(1-348)/ARNT2(1-439) and mouse NPAS4(1-329)/ARNT2(1-481)) from baculovirus infected Sf9 insect cells or human AhR(1-287)/ARNT(1-364) from bacteria. Seven purified homo- or heterodimer complexes were incubated with SELEX libraries (Random 18mer or FixedCore (8N-CGTG-10N) 22mer) and bound DNA were separated by electrophoretic mobility assay (EMSA), followed by DNA isolation and PCR amplification. Random 18mer libraries were subjected to 3 rounds of SELEX, whereas FixedCore (8N-CGTG-10N) 22mer libraries were subjected to 1 or 2 rounds of SELEX. The Random 18mer initial library and round 3 were sequenced using a NextSeq500 1x75bp HighOutput mode yielding ~30million reads per sample. The FixedCore (8N-CGTG-10N) 22mer initial library, round 1 and round 2 were sequenced using a HighSeq2500 yielding ~30million reads per sample.
 
Contributor(s) Bersten DC, Whitelaw ML
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Submission date Oct 23, 2020
Last update date Jul 02, 2022
Contact name David C Bersten
E-mail(s) david.bersten@adelaide.edu.au
Phone 0403159545
Organization name The University of Adelaide
Department Molecular and Biomedical Sciences
Lab Bersten Lab
Street address Victoria Drive
City Adelaide
State/province SA
ZIP/Postal code 5001
Country Australia
 
Platforms (2)
GPL19424 Illumina NextSeq 500 (synthetic construct)
GPL19604 Illumina HiSeq 2500 (synthetic construct)
Samples (11)
GSM4852178 Random 18mer library
GSM4852179 HIF1a ARNT round 3
GSM4852180 HIF2a ARNT round 3
Relations
BioProject PRJNA671496
SRA SRP288384

Download family Format
SOFT formatted family file(s) SOFTHelp
MINiML formatted family file(s) MINiMLHelp
Series Matrix File(s) TXTHelp

Supplementary file Size Download File type/resource
GSE159989_processed_data.tar.gz 462.0 Mb (ftp)(http) TAR
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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