Curation Information

Publication
Genome-scale analyses of Escherichia coli and Salmonella enterica AraC reveal noncanonical targets and an expanded core regulon.;Stringer AM, Currenti S, Bonocora RP, Baranowski C, Petrone BL, Palumbo MJ, Reilly AA, Zhang Z, Erill I, Wade JT;Journal of bacteriology 2014 Feb; 196(3):660-71 [24272778]
TF
AraC [A0A0F6AWN9, view regulon]
Reported TF sp.
Salmonella enterica subsp. enterica serovar Typhimurium str. 14028S
Reported site sp.
Salmonella enterica subsp. enterica serovar Typhimurium str. 14028S
Created by
Erill Lab
Curation notes
-
External databases
ArrayExpress (E-MTAB-1915) http://www.ebi.ac.uk/arrayexpress/arrays/E-MTAB-1915/. ArrayExpress (E-MTAB-1901) http://www.ebi.ac.uk/arrayexpress/arrays/E-MTAB-1901/.

Experimental Process

ChIP-seq was performed on cells growing on LB or LB+arabinose. Chipseq results were validated with ChIP-qPCR. Motif discovery was performed in the ChIP enriched regions and a motif matching the known consensus was identified. RNAseq was also used to analyze expression patterns on w-t and AraC mutants in presence or absence of arabinose. Expression changes >4 fold between w-t and mutant upon arabinose were deemed significant. The conservation of identified sites was evaluated with multiple sequence alignments against several related species.

ChIP assay conditions
S. enterica: 40 ml cells expressing C-terminally FLAG-tagged AraC (CB005; Table 3) were grown in LB + 0.2% arabinose at 37 °C to an OD 600 of 0.6-0.8.
ChIP notes
Cells were crosslinked for 20 minutes with formaldehyde (1% final concentration), pelleted by centrifugation and washed once with Tris-buffered saline (TBS). Cell pellets were resuspended in 1 ml FA lysis buffer (50 mM Hepes-KOH, pH 7, 150 mM NaCl, 1 mM EDTA, 1% Triton X- 100, 0.1% sodium deoxycholate, 0.1% SDS) with 2 mg/ml lysozyme and incubated at 37 °C for 30 minutes. Samples were then chilled and sonicated for 30 minutes in a Bioruptor sonicator (Diagenode) with 30 s on/30 s off pulsing at maximum amplitude. Samples were pelleted in a microcentrifuge to remove debris and supernatants (“chromatin”) were saved, 1 ml FA lysis buffer was added, and samples were stored indefinitely at -20 °C. For each immunoprecipitation (IP), 500 μl chromatin was incubated with 300 μl FA lysis buffer, 20 μl Protein A Sepharose slurry (50%) in TBS and either 1 μl anti-β (RNA polymerase subunit) antibody (NeoClone) or 2 μl M2 anti-FLAG antibody (Sigma) for 90 minutes at room temperature with gentle mixing on a Labquake Rotisserie Rotator (Thermo Scientific). For ChIP of AraC-TAP, Protein A sepharose and antibody was replaced with IgG sepharose. Beads were then pelleted at 1,500 x g in a microcentrifuge for 1 minute. The supernatant was removed and the beads were resuspended in 750 μl FA lysis buffer and transferred to a Spin-X column (Corning). Beads were then incubated for 3 minutes with gentle mixing on a rotisserie rotator before being pelleted at 1,500 x g in a microcentrifuge for 1 minute. Equivalent washes were performed with FA lysis buffer, high salt FA lysis buffer (50 mM Hepes-KOH, pH 7, 500 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS), ChIP wash buffer (10 mM Tris-HCl, pH 8.0, 250 mM LiCl, 1 mM EDTA, 0.5% Nonidet-P40, 0.5% sodium deoxycholate) and TE (10 mM Tris-HCl, pH 7.5, 1 mM EDTA). After the TE wash, beads were transferred to a fresh Spin-X column and eluted with 100 μl ChIP elution buffer (50 mM Tris-HCl, pH 7.5, 10 mM EDTA, 1% SDS) for 10 minutes at 65 °C with occasional agitation. Eluted samples were centrifuged at 1,500 x g in a microcentrifuge for 1 minute. Supernatants were decrosslinked by boiling for 10 minutes and cleaned up using a PCR purification kit (Qiagen). Further wash steps involved incubation of samples with the wash solution for 3 min with gentle mixing on a rotisserie rotator before pelleting beads at 1,500 x g in a microcentrifuge for 1 min. Following the 90 min incubation, beads were washed twice with FA lysis buffer and twice with 10 mM Tris-HCl, pH 7.5. Beads were resuspended in 100 μL 1X Quick Blunting Buffer (NEB) containing dNTPs (concentration specified by NEB Quick Blunting kit) and 1 μL Quick Blunting enzyme mix (NEB), and incubated at room temperature for 30 min with gentle mixing. Beads were washed twice with FA lysis buffer and twice with 10 mM Tris-HCl, pH 8.0. Beads were resuspended in 100 μL Buffer 2 (NEB) containing 2 mM dATP and 2 μL Klenow DNA polymerase (NEB), and incubated for 30 min at 37 °C with gentle mixing. Beads were washed twice with FA lysis buffer and twice with 10 mM Tris-HCl, pH 7.5. Barcoded adapter oligonucleotides JW2870 + JW2881 or JW2876 + JW2887 (two different barcoded pairs) were annealed by boiling a mixture of 100 μM each in 10 mM Tris, pH 7.5, 50 mM NaCl, 1 mM EDTA, and cooling slowly. The adapter oligonucleotide mix was then diluted 10-fold in water. Beads were resuspended in 1X DNA Quick Ligase buffer (NEB), 1 μL adapter oligonucleotide mix and 2 μL Quick DNA Ligase (NEB), and incubated at room temperature for 15 min with gentle mixing. Beads were washed twice with FA lysis buffer, and once each with high salt FA lysis buffer, ChIP wash buffer and TE. After the TE wash, beads were transferred to a fresh Spin-X column and eluted with 100 μl ChIP elution for 10 minutes at 65 °C with occasional agitation. Samples were decrosslinked by boiling for 10 minutes. DNA was extracted with phenol/chlorofom/isoamyl alcohol, ethanol precipitated, and resuspended in 11 μL H 2O. 1 μL DNA was used for real time PCR amplification with oligonucleotides JW1169 + JW2327 using an ABI 7500 Fast real time PCR machine. The number of cycles required to reach a Delta Rn score of 0.1 was recorded (“X”). 8 μL of the remaining DNA was amplified using conventional PCR with oligonucleotides JW1169 + JW2327 for X+3 cycles with an annealing temperature of 60 °C. PCR products were purified using Ampure XP magnetic beads (Ampure) and resuspended in 20 μL H2O. Purified PCR products between 200 bp and 600 bp were gel purified from an 8% non-denaturing polyacrylamide gel, eluted overnight in 0.4 M NaCl, and ethanol precipitated to generate the final ChIP-seq libraries. Libraries were resuspended in 10 μL H2O and quantified using a Qubit fluorimeter (Invitrogen). Libraries were sequenced using a HiSeq 2000 sequencer (Illumina; University at Buffalo Next Generation Sequencing Core Facility). Sequence reads were aligned to non-repetitive sequences in the S. enterica subsp. enterica serovar Typhimurium 14028s genome using the CLC Genomics Workbench and overall coverage was determined using custom Python scripts. AraC-bound regions were called for any genomic coordinate with >200 sequence reads mapping to both strands. ChIP-seq peaks were identified as the position with the most number of mapped sequence reads within each AraC- bound region.

Transcription Factor Binding Sites


GCGAATAATTACCGCCATC
CGTAGCCATTTAATCCATA
CATAGCATAATAATGAATA
ATTAGCATTTTTGTCCATA
TATGGCTGGAATGTCCACA
GCGAATAATTACCGCCATC
CGTAGCCATTTAATCCATA
CATAGCATAATAATGAATA
ATTAGCATTTTTGTCCATA
TATGGCTGGAATGTCCACA
TGAGACTTCTCCCCATCAGATGATGGCGGTAATTATTCGCCTGCGAGCTTTTTTTTGCGTAGCAGGATCACGCCGCTGATATGGATAAAATGGTTATACGT None

Gene Regulation

Regulated genes for each binding site are displayed below. Gene regulation diagrams show binding sites, positively-regulated genes, negatively-regulated genes, both positively and negatively regulated genes, genes with unspecified type of regulation. For each indvidual site, experimental techniques used to determine the site are also given.

Site sequence Regulated genes Gene diagram Experimental techniques TF function TF type
GCGAATAATTACCGCCATC STM14_0178, STM14_0177,
... ... STM14_0178 STM14_0177 STM14_0179
Experimental technique details ChIP-PCR (ECO:0005620) - Experimental technique details ChIP-Seq (ECO:0006009) - Experimental technique details Motif-discovery (ECO:0005558) - Experimental technique details Multiple sequence alignment (MSA) (ECO:0005556) - Experimental technique details RNA-Seq (ECO:0005664) - activator monomer
CGTAGCCATTTAATCCATA araE, ygeA
... ... araE ygeA
Experimental technique details ChIP-PCR (ECO:0005620) - Experimental technique details ChIP-Seq (ECO:0006009) - Experimental technique details Motif-discovery (ECO:0005558) - Experimental technique details Multiple sequence alignment (MSA) (ECO:0005556) - Experimental technique details RNA-Seq (ECO:0005664) - activator monomer
CATAGCATAATAATGAATA araJ
... ... araJ
Experimental technique details ChIP-PCR (ECO:0005620) - Experimental technique details ChIP-Seq (ECO:0006009) - Experimental technique details Motif-discovery (ECO:0005558) - Experimental technique details Multiple sequence alignment (MSA) (ECO:0005556) - Experimental technique details RNA-Seq (ECO:0005664) - activator monomer
ATTAGCATTTTTGTCCATA araB, araA, araD, araC,
... ... araB araA araD araC yabI
Experimental technique details ChIP-PCR (ECO:0005620) - Experimental technique details ChIP-Seq (ECO:0006009) - Experimental technique details Motif-discovery (ECO:0005558) - Experimental technique details Multiple sequence alignment (MSA) (ECO:0005556) - Experimental technique details RNA-Seq (ECO:0005664) - activator monomer
TATGGCTGGAATGTCCACA araB, araA, araD, araC,
... ... araB araA araD araC yabI
Experimental technique details ChIP-PCR (ECO:0005620) - Experimental technique details ChIP-Seq (ECO:0006009) - Experimental technique details Motif-discovery (ECO:0005558) - Experimental technique details Multiple sequence alignment (MSA) (ECO:0005556) - Experimental technique details RNA-Seq (ECO:0005664) - activator monomer