Curation Information

Publication

ChIP analysis unravels an exceptionally wide distribution of DNA binding sites for the NtcA transcription factor in a heterocyst-forming cyanobacterium.;Picossi S, Flores E, Herrero A;BMC genomics 2014 Jan 13; 15(.):22 [24417914]

TF

NtcA [P0A4U6, view regulon]

Reported TF sp.

Nostoc sp. PCC 7120

Reported site sp.

Nostoc sp. PCC 7120

Created by

Erill Lab

Curation notes

-

External databases

NCBI GEO (GSE51865) http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE51865.

Experimental Process

ChIP seq with NtcA antibody was performed on Anabaena sp. PCC 7120 cells in a N-depleted medium. CysFinder was applied for motif discovery in the whole dataset. ChIP-qPCR was used to validate ChIP-seq on a control gene. Several bound regions were verified in vitro through EMSA.

ChIP assay conditions

Wild-type Anabaena sp. PCC 7120 cells growing in bubbled cultures with ammonium as the N source were subjected to incubation in a combined N-depleted medium for 3 hours, after which the cultures were treated with formaldehyde to fix the proteins bound to DNA. After cell lysis and DNA fragmentation, the extracts were treated with an anti-NtcA antibody to specifically immunoprecipitate the NtcA-bound DNA. The immunoprecipitated material was then incubated at 65ºC to reverse the crosslinking, and the DNA was isolated. A sample of total DNA was also isolated prior to anti-NtcA treatment of the extracts to serve as the control input sample. Cells of Anabaena sp. (also known as Nostoc sp.) strain PCC 7120 growing exponentially (3-5 μg Chl/ml) in the light (75 μE·m-2·s-1) at 30°C in BG110 medium supplemented with 10 mM NaHCO3 (referred to as BG110C) containing 6 mM NH4Cl and 12 mM TES and bubbled with a mixture of air + 1% CO2 were collected, washed with BG110C, resuspended in BG110C, and incubated in the same conditions for 3 h. Formaldehyde was then added to the cultures to a final concentration of 1%, and the cultures were incubated for 15 min (no aeration, occasional shaking). Glycine was added at 125 mM final concentration and the incubation was continued for 5 min to stop the fixing reaction. The cells were then filtered, washed with cold TBS (20 mM Tris–HCl, pH 7.4, 140 mM NaCl) and collected in tubes (25 ml of culture per tube). The pellets were frozen in liquid nitrogen and stored at -20°C until used.

ChIP notes

Cells corresponding to about 150 ml of culture (6 tubes) were used for each ChIP experiment. Pellets corresponding to about 25 ml of culture were resuspended in 500 μl of lysis buffer (50 mM HEPES/KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, supplemented with Mini EDTA-free protease inhibitor cocktail [Roche]). Cells were supplemented with 150 μl of glass beads (acid-washed, 425-600 μm [Sigma]) and broken in a multivortexer at 2000 rpm for 1 h at 4°C. The cell lysates were collected by centrifugation and the extracts were subjected to sonication to shear the DNA into about 200-bp fragments (40 cycles of 10s, 30s ice, 10% amplitude, in a Branson Digital Sonifier). After centrifugation to eliminate cell debris, the whole-cell extracts were stored at -20°C or immediately used for immunoprecipitation. Immunoprecipitation of DNA was carried out as described in Hanaoka and Tanaka, with some modifications. Whole-cell extracts were prepared at 4 mg/ml of total protein with lysis buffer (in 500 μl total volume). A 50-μl sample was taken as the input sample, and the extracts were pre-treated with 0.6 mg of lysis-buffer-equilibrated Dynabeads Protein G (Invitrogen) (to avoid non-specific binding of DNA to the Protein G). Anti-NtcA antibody (or H2O for the mock sample) was added and incubated at 4°C with rotation overnight. The extracts were treated with 0.6 mg of Dynabeads Protein G for 2 h at 4°C with rotation. The Dynabeads were washed twice with 1.5 ml of lysis buffer (5 min, rotation), and once with 1.5 ml each buffer 1 (lysis buffer containing 500 mM NaCl), buffer 2 (10 mM Tris–HCl, pH 8, 250 mM LiCl, 0.5% NP-40, 1 mM EDTA), and buffer 3 (10 mM Tris–HCl, pH 7.5, 1 mM EDTA, 50 mM NaCl). The Dynabeads were resuspended in a solution of DNase-free RNase A (0.2 μg/μl in TE), incubated for 30 min at 37°C, and washed with 1.5 ml wash buffer 3. To elute the immunoprecipitated material, the Dynabeads were resuspended in 50 μl of elution buffer (50 mM Tris–HCl, pH 7.5, 10 mM EDTA, 1% SDS) and incubated at 65°C for 30 min. The elution step was repeated once and the two eluates were combined. For crosslinking reversion, the eluted material was incubated at 65°C for 5 h. The input sample was processed in parallel (Tris–HCl, pH 7.5, EDTA and SDS was added to reach the same concentration as in the ChIP sample). To eliminate proteins, Proteinase K was added at 0.4 μg/μl (final concentration) and the mixture was incubated for 1 h at 55°C. DNA was purified by phenol/chloroform/isoamyl alcohol extraction (25:24:1) followed by two extractions with chloroform/isoamyl alcohol (24:1). DNA was ethanol-precipitated using ammonium acetate and glycogen, and the pellet was washed twice with 70% ethanol, air-dried and resuspended in 25 μl purified H2O. For ChIP-Seq DNA samples, this protocol was repeated three times using cells from independent inductions, and the resulting DNA was mixed together and concentrated to 25 μl. Input and ChIP DNA samples were sent for sequencing at the Functional Genomics Core Facility of the Institute for Research in Biomedicine, Barcelona (Spain) (Herbert Auer). Next generation sequencing was carried out using Illumina’s sequencing technology. ChIP DNA Sample Prep Kit (Illumina) was used for library preparation. Libraries were loaded at 8 pM concentration into the flow cell using the Cluster Station running recipe V7 with the Single-Read Cluster Generation Kit v4 (all Illumina). The flow cell was loaded into the Genome Analyzer II and samples were sequenced for 120 nucleotides from a single end using the Sequencing Kit v5 and recipe v8 (all Illumina). Manufacturer’s recommendations were strictly followed. Illumina sequencing data were pre-processed with the standard Illumina pipeline version 1.5 and sequences were aligned to the Anabaena sp. PCC 7120 genome (http://genome.microbedb.jp/cyanobase/Anabaena webcite) with the Bowtie software 0.12.5. The percentage of reads mapped to the genome was 92.3% for the Input sample (HQ reads: 30,192,934, 64.9% of total) and 94.2% for the ChIP sample (HQ reads: 31,352,138, 68.5% of total). The analysis of the results was carried out using the Triform algorithm method (Karl Kornacker). For detected double-strand peak regions, the peak locations were reported as the averages of the forward and reverse peak locations; the z-scores were calculated according to equations (4) - (6) , with C (x) being replaced by the sum of the coverages on the forward and reverse peak locations; and the associated discrete p-values were adjusted for multiple testing by application of the Tarone-modified distribution-free Benjamini-Yekutieli method, similar to a method recommended in Gilbert, 2005 . The Q value measures the statistical significance of the peak identifying the target region, defined as the estimated false discovery rate (FDR) among the rows whose Q value is no larger than a chosen FDR. The NLQ value is defined as the -log10(Q value).

Transcription Factor Binding Sites

• Sites reported in the paper
• Sites matched in the genome
• Site-specific quantitative values
• High-throughput data

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.