Open Access Research article

Combined ChIP-Seq and transcriptome analysis identifies AP-1/JunD as a primary regulator of oxidative stress and IL-1β synthesis in macrophages

Richard P Hull1, Prashant K Srivastava1, Zelpha D’Souza1, Santosh S Atanur1, Fatima Mechta-Grigoriou2, Laurence Game1, Enrico Petretto1, H Terence Cook3, Timothy J Aitman1 and Jacques Behmoaras3*

Author Affiliations

1 MRC Clinical Sciences Centre, Imperial College London, Hammersmith hospital, Du Cane Road W12 0NN, London, UK

2 Stress and Cancer Laboratory, Institut Curie, 26 Rue d’Ulm, Paris, France

3 Centre of Complement and Inflammation Research, Imperial College London, Du Cane Road W12 0NN, London, UK

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BMC Genomics 2013, 14:92  doi:10.1186/1471-2164-14-92

Published: 11 February 2013

Additional files

Additional file 1: Figure S1:

Genome-wide expression analysis in basal and LPS stimulated BMDMs. Genome wide expression analysis by microarrays was performed in BMDMs transfected with rat Jund or scrambled control siRNA for the unstimulated condition (A) or following eight hours of LPS stimulation (B) in WKY BMDMs and over an eight hour time course of LPS stimulation in WKY and WKY.LCrgn2 BMDMs (C). Heat maps of hierarchically clustered significantly differentially expressed genes (<5% FDR threshold) are displayed. All experiments were performed in 4 biological replicates for each strain or siRNA transfected. Figure S2. Validation of microarray data between WKY and WKY.LCrgn2 BMDMs over an eight hour LPS stimulation timecourse. Validation of microarray data by qRT-PCR. Samples were amplified using a set of four biological replicates with three technical replicates per sample. Relative gene expression was measured by qRT-PCR and normalised with Hprt for WKY and WKY.LCrgn2 BMDMs. *P<0.05; **P<0.01;***P<0.001 statistically significantly different to WKY using a two way ANOVA to compare the overall timecourse with Bonferonni’s post-tests to compare individual time points. Figure S3. ChIP-Seq peak validations by ChIP-qPCR. ChIP-Seq peaks identified at a posterior probability threshold of 0.9 for basal WKY BMDMs were validated by qPCR (A) and for LPS stimulated WKY BMDMs (B) and WKY.LCrgn2 BMDMs peaks (C). Samples were amplified using a set of biological triplicates with three technical replicates per sample. Results expressed as mean fold change over IgG. **P<0.01, *P<0.05, ns; non-significant using a paired t-test (one-tailed) to compare whether % input for the JunD ChIP qPCR was significantly different to % input for IgG. Figure S4.Il1b and Prkca confirmed as primary JunD targets by qPCR validation. The aligned reads comprising peak passing the posterior probability threshold of 0.9 for each JunD-bound gene in the WKY strain in the basal state for l1b (A) and the LPS stimulated state for Prkca (B) are shown in genome browser views along with the peak in the WKY.LCrgn2 strain. Samples from WKY and WKY.LCrgn2 strains were amplified using three biological replicates with three technical replicates per sample. Results expressed as mean fold change over IgG. *P<0.05; **P<0.01; using a one-tailed unpaired t-test to detect statistically significant differences between the strain and condition pairs. Error bars represent standard error of the mean. Figure S5. Integrative analysis identifies the transcription factor Bcl2l11 as a primary JunD target. Jund microarray-determined expression patterns in WKY and WKY.LCrgn2 BMDMs over an eight hour LPS timecourse using four biological replicates per strain were used for Spearman correlation analysis (A) with the rest of the transcripts on the microarrays. The expression of Bcl2l11 (B) was significantly correlated to the Jund expression pattern (Spearman correlation 0.9, corrected p-value=8.6x10-5). Significant differential expression of the gene was seen following siRNA knockdown of Jund (C). Fold changes are of control siRNA versus Jund siRNA expression. The positive fold change indicates higher expression in BMDMs transfected with scrambled control siRNA i.e. with a higher level of Jund expression compared to Jund siRNA. Abbreviations: Chr.; chromosome, FDR: false discovery rate. Three JunD binding events were identified at a posterior probability threshold of 0.9 in LPS stimulated WKY BMDMs (D) located in the gene promoter and second intron.

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Additional file 2: Table S1:

Validation of differentially expressed genes identified by siRNA microarray data analysis with quantitative PCR. Table S2. Sequencing and mapping statistics for ChIP-Seq in WKY and WKY.LCrgn2 BMDMs. Table S3. Gene ontology analysis of JunD-bound genes in basal WKY BMDMs. Table S4. Gene ontology analysis of JunD-bound genes in basal WKY.LCrgn2 BMDMs. Table S5. Gene ontology analysis of JunD-bound genes in LPS stimulated WKY.LCrgn2 BMDMs. Table S6. Gene ontology analysis of JunD-bound genes in LPS stimulated WKY BMDMs. Table S7. Sequences of the four individual siRNAs that comprise siGENOME SMARTpool M-092127-00-0010 (Dharmacon). Table S8. Primer sequences used for qRT-PCR validation of microarray data. Table S9. Primer sequences used for qPCR validation of ChIP-Seq data.

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