Gene Expression Microarrays. For microarrays, purity of the RNA was further assessed by

Transcription

1 Supplemental Methods Gene Expression Microarrays. For microarrays, purity of the RNA was further assessed by an Agilent 2100 Bioanalyzer. 500 ng of RNA was reverse transcribed into crna and biotin-utp labeled using the Illumina TotalPrep RNA Amplification Kit (Ambion). crna was quantified using an Agilent Bioanalyzer 2100 and hybridized to the Illumina mouserefseq-8v2 Expression BeadChip using standard protocols. Image data was converted into un-normalized Sample Probe Profiles using the Illumina BeadStudio software and analyzed on the VAMPIRE microarray analysis framework 3. Stable variance models were constructed for each of the experimental conditions (n=2). Differentially expressed probes were identified using the unpaired VAMPIRE significance test with a 2-sided, Bonferroni-corrected threshold of Bonf = The VAMPIRE statistical test is a Bayesian statistical method that computes a modelbased estimate of noise at each level of gene expression. This estimate was then used to assess the significance of apparent differences in gene expression between 2 experimental conditions. Lists of altered genes generated by VAMPIRE were mapped to pathways using the VAMPIRE tool GOby to determine whether any KEGG categories were overrepresented using a Bonferroni error threshold of Bonf = Gene function analysis was performed using KEGG pathways classified into broader functional groups as shown in Supplemental Tables 1-6. Gene expression microarray data is deposited at Gene Expression Omnibus (GEO), accession # GSE Chromatin immunoprecipitation. Following fixation, nuclei were isolated, lysed in buffer containing 1% SDS, 10 mm EDTA, 50 mm Tris-HCl ph 8.0, and protease inhibitors, and sheared with a Diagenode Bioruptor to chromatin fragment sizes of base pairs. Chromatin was immunoprecipitated with antibodies to Bcl-6, pre-immune IgG, p300, Hdac3 (Santa Cruz Bio), acetylated histone H3 (Millipore), p65 (Santa Cruz and Abcam), or H3K4me1, H3, and H4 (AbCam).

2 ChIP-seq. Short DNA reads were aligned against the mouse mm9 reference genome using the Illumina Pipeline Suite v1.4. Standard ELAND parameters were used that allow up to 2 mismatches in the first 32 bases of the read. Data analysis was performed using HOMER, a software suite for ChIP-seq analysis. The methods, which are described below, have been implemented and are freely available at Only tags that mapped uniquely to the genome were considered for further analysis. ChIP-seq experiments were visualized by preparing custom tracks for the UCSC Genome browser in a manner similar to that described elsewhere (Robertson et al. 2007). Identification of ChIP-seq Peaks. Since each ChIP-seq tag represents the edge of a ChIP fragment, we adjusted the position of each tag 3 of its position by 75 bp, corresponding to half the recommended fragment length for Illumina sequencing. We considered one tag from each unique position to eliminate peaks resulting from clonal amplification of fragments during the ChIP-seq protocol. Peaks (binding sites) were identified by searching for clusters of tags within a sliding 200 bp window, requiring adjacent clusters to be at least 1 kb away from each other. The threshold for the number of tags that determine a valid peak was selected for a false discovery rate of <0.01, as empirically determined by repeating the peak finding procedure using randomized tag positions. We also required peaks to have at least 4-fold more tags (normalized to total count) than input or IgG control samples. In addition, we required 4-fold more tags relative to the local background region (10 kb) to avoid identifying regions with genomic duplications or non-localized binding. Peaks were annotated to gene products by identifying the nearest RefSeq TSS. Analysis of ChIP-seq Peaks using HOMER. Peak sequences from +/- 100bp relative to the peak center were compared to 50,000 randomly selected genomic fragments of the same size and matched for CpG% to remove sequence bias introduced by CpG Islands. Motif discovery was performed using a comparative algorithm similar to those previously described (Segal et al.

3 2002; Linhart et al. 2008) and an in depth description will be published elsewhere (Benner et al., in preparation). Briefly, sequences were divided into target and background sets for each application of the algorithm. Motifs of length 8, 10, and 12 bp were identified separately by first exhaustively screening all oligos for enrichment in the target set compared to the background set using the cumulative hypergeometric distribution to score enrichment. Up to two mismatches were allowed in each oligonucleotide sequence to increase the sensitivity of the method. The top 100 oligonucleotides of each length with the lowest P-values were then converted into probability matrices and heuristically optimized to maximize hypergeometric enrichment of each motif in the given data set. As optimized motifs were found they were removed from the data set to facilitate the identification of additional motifs. Sequence logos were generated using WebLOGO ( ChIP-seq data is deposited at Gene Expression Omnibus (GEO), accession # GSE Supplemental References Linhart, C., Halperin, Y., and Shamir, R Transcription factor and microrna motif discovery: the Amadeus platform and a compendium of metazoan target sets. Genome research 18(7): Robertson, G., Hirst, M., Bainbridge, M., Bilenky, M., Zhao, Y., Zeng, T., Euskirchen, G., Bernier, B., Varhol, R., Delaney, A. et al Genome-wide profiles of STAT1 DNA association using chromatin immunoprecipitation and massively parallel sequencing. Nature methods 4(8): Segal, E., Barash, Y., Simon, I., Friedman, N., and Koller, D From promoter sequence to expression: a probabilistic framework. Proc 6th Inter Conf on Research in Computational Molecular Biology (RECOMB), Washington, DC.

4 Supplemental Figures and Tables Supplemental Fig. 1. Bcl-6 co-regulates the Tlr-4 elicited gene expression program and is a target of Tlr-4 signaling. (A) Comparison of genes induced or repressed by exposure to LPS (100 ng/ml) for 6 hours in Bcl-6 +/+ (WT) versus Bcl-6 -/- (KO) BMDMs. (B) qpcr of microarrayidentified Bcl-6 regulated genes in WT and KO BMDMs at 0, 2, or 6 hours following exposure to LPS (100 ng/ml). Mean relative expression levels compared to WT BMDMs at baseline (0 hr) + SD are listed. (C) Expression of Bcl-6 mrna in BMDMs exposed to LPS 100 ng/ml for 0, 2, or 6 hours. Altered expression versus baseline (0 hr) determined using 2-tailed t-tests. (D) Levels of Bcl-6 protein versus -actin in BMDMs exposed to LPS 100 ng/ml for 0, 2, or 6 hours. Relative quantifications are listed. (E), BMDMs were pre-treated for 30 minutes with control solvent or inhibitors to Mek1/2 (U0126, 10 M), Erk (Erk Inhibitor II, 1 M), PI3 kinase (wortmannin, 100 nm) or NF- B (Bay , 10 M) and assessed for expression of Bcl-6 and Ccl2 with or without further exposure to LPS (100 ng/ml) for 2 hours. Groups compared for statistical analysis are shown in brackets. Statistical testing using one-way ANOVA with Tukey s multiple comparison tests. For B, C, and E values are expressed as means + SD. Statistical significance indicated: + p < 0.05, # p < 0.01, * p < Supplemental Fig. 2. Chromatin immunoprecipitation sequencing reveals extensive colocalization of Bcl-6 with NF- B. (A) ChIP qpcr at ChIP-seq-identified Bcl-6 binding sites using Bcl-6 and control IgG antibody in WT versus KO BMDMs. Enrichment is expressed as mean % of input chromatin + SD. Statistical testing to compare enrichment with Bcl-6 antibody using 2-tailed t-tests: + p<0.05, # p < 0.01, * p < (B, C) Distributions of Bcl-6 binding sites in unstimulated (B) or LPS-stimulated (C) BMDMs relative to transcription start sites. (D, E) Motif analysis of Bcl-6 ChIP-sequenced DNA in unstimulated (D) or LPS-stimulated (E) WT BMDMs.

5 (F) ChIP qpcr in WT BMDMs over a 12-hour time course of LPS stimulation using Bcl-6 or control IgG antibodies at ChIP-seq-identified Bcl-6 binding sites in unstimulated cells (Csf1 and Ccl2) or LPS stimulated cells (Ccl2 and Ifitm1). The position of the Bcl-6 binding site relative to the transcription start site is listed for each. Values are expressed as means of % of input chromatin + SD. Statistical testing using 2-tailed t-tests: + p < 0.05, # p < 0.01, * p < Supplemental Fig. 3. Assessment of NF- B cistromic regulation. (A, B) Motif analysis of NF- B p65 ChIP-sequenced DNA in unstimulated (A) or LPS-stimulated (B) WT BMDMs. (C), ChIP sequencing in unstimulated versus LPS stimulated BMDMs at the Bcl-6 locus shows inducible NF- B p65 binding at several up- and downstream locations. (D) Venn diagram comparing the cistromes of Bcl-6 in unstimulated (Bcl-6 unstimulated) or LPS stimulated (Bcl-6 + LPS) BMDMs versus the NF- B p65 cistrome in LPS stimulated cells (NF- B p65 + LPS). The numbers of overlapping and non-overlapping genes are listed for each. Supplemental Fig. 4. ChIP and ChIP-sequencing reveal Bcl-6 and NF- B co-regulation of inflammatory genes. (A) ChIP sequencing tracks for acetylated histone 3 (ach3), monomethylated histone 3 lysine 4 (H3K4me1), and RNA polymerase II (RNA pol II) in unstimulated BMDMs as well as p300, Pu.1, NF- B p65, and Bcl-6 in unstimulated or LPS stimulated (100 ng/ml for 3 hours) BMDMs along the Ccl2/Ccl7/Ccl11 gene cluster. For factors sequenced with and without exposure to LPS, track heights were normalized to the number of aligned reads. (B, C), ChIP qpcr assessment of p65 and p300 occupancy at proximal Bcl-6 / NF- B binding sites expressed as mean percentage of input chromatin. Values are expressed as means + SD. Statistical testing using one-way ANOVA with Tukey s multiple comparison tests, + p < 0.05, # p < 0.01, * p < Significant differences are noted relative to unstimulated

6 WT BMDMs. Supplemental Fig. 5. Epigenetic co-regulation of inflammatory genes by Bcl-6 and NF- B. (A, B) ChIP qpcr of relative monomethylated histone 3K4 (H3K4me1) and H3K4me1 normalized to histone 3 (H3) enrichment in WT and KO BMDMs with or without exposure to LPS (100 ng/ml for 3 hours) at Bcl-6/p65 binding sites. (C, D) ChIP qpcr of relative acetylated histone 3 (AcH3) and total histone 3 (H3) in WT and KO BMDMs with or without exposure to LPS (100 ng/ml for 3 hours) at Bcl-6/p65 binding sites. The annotated gene name and position of the Bcl-6 binding site relative to the transcription start site is listed. 36b4 is a negative control DNA region lacking a Bcl-6 or p65 binding site. Values are expressed as means + SD. Statistical testing using one-way ANOVA with Tukey s multiple comparison tests, + p < 0.05, # p<0.01, * p < Significant differences are noted relative to unstimulated WT BMDMs. Supplemental Table 1. Functional categorization of genes controlled by Bcl-6 in macrophages. Supplemental Table 2. Functional categorization of genes co-regulated by Bcl-6 and LPS in macrophages. Supplemental Table 3. Functional categorization of genes annotated with Bcl-6 binding sites in quiescent wild type bone marrow macrophages. Supplemental Table 4. Functional categorization of genes annotated with Bcl-6 binding sites in LPS stimulated wild type bone marrow macrophages. Supplemental Table 5. Functional categorization of genes annotated with NF- B p65 binding sites in LPS stimulated wild type bone marrow macrophages.

7 Supplemental Table 6. Functional categorization of genes annotated with proximal Bcl-6 and NF- B p65 binding sites in wild type bone marrow macrophages. Supplemental Table 7. Primers used for gene expression analysis with qpcr. Supplemental Table 8. Primers used for ChIP qpcr.