Conceived and designed the experiments: HCM CHL DMS. Performed the experiments: CHL DMS. Analyzed the data: DMS HCM. Contributed reagents/materials/analysis tools: HCM. Wrote the paper: DMS HCM.
Current address: Center for Reproductive Medicine, Good Moonhwa Hospital, S&M Research Institute, S&M Incoporated, Busan, South Korea
The authors have declared that no competing interests exist.
IRF8 (Interferon Regulatory Factor 8) is a transcription factor expressed throughout B cell differentiation except for mature plasma cells. Previous studies showed it is part of the transcriptional network governing B cell specification and commitment in the bone marrow, regulates the distribution of mature B cells into the splenic follicular and marginal zone compartments, and is expressed at highest levels in germinal center (GC) B cells. Here, we investigated the transcriptional programs and signaling pathways affected by IRF8 in human and mouse GC B cells as defined by ChIP-chip analyses and transcriptional profiling. We show that IRF8 binds a large number of genes by targeting two distinct motifs, half of which are also targeted by PU.1. Over 70% of the binding sites localized to proximal and distal promoter regions with ∼25% being intragenic. There was significant enrichment among targeted genes for those involved in innate and adaptive immunity with over 30% previously defined as interferon stimulated genes. We also showed that IRF8 target genes contributes to multiple aspects of the biology of mature B cells including critical components of the molecular crosstalk among GC B cells, T follicular helper cells, and follicular dendritic cells.
IRF8, one of nine members of the IRF family of transcription factors, functions in modulating immune responses and as a central element in the IFN signaling cascade. The gene is constitutively expressed in macrophages where it has been identified at the promoter regions of a large number of genes critical to macrophage differentiation and function
Studies of IRF8-deficient mice also identified critical roles in dendritic cell (DC) development and function. IRF8 KO mice lack plasmacytoid DCs (pDC) and CD11c+CD8α+ DCs
Early on, it was shown that IRF8 is constitutively expressed by normal mouse B cells and lymphoma cell lines with features of pro-B and pre-B cells but not by plasmacytomas, tumors of mature plasma cells
Additional studies showed that among mouse and human B lineage cells IRF8 is expressed at the highest levels in germinal center (GC) B cells and lymphomas of GC origin but is extinguished in terminally differentiated plasma cells and plasma cell neoplasms
To identify direct transcriptional targets for IRF8 in human GC B cells, we hybridized IRF8-bound chromatin obtained by ChIP from three cell lines of GC origin (ODH1, VAL and LY1) to Nimblegen promoter tiling arrays consisting of probes covering 3.5 kb upstream to 0.75 kb downstream of transcriptional start sites (TSS); a multiple myeloma cell line (MMS1) with very little or no expression of IRF8 served as a negative control. The number of genes identified as IRF8-bound in the three GC lines were 1,563 for VAL, 1,724 for ODH1 and 2790 for LY1 with 271 genes being common to all three lines (
Labeled IRF8 ChIP samples and input samples from the three human cell lines of GC B cell origin were applied to Nimblegen HG18 385 k arrays and peak signals were analyzed by sliding window algorithm with threshold of FDR<0.01. (A) Venn diagram of IRF8 targets in three cell lines. Numbers in parentheses indicate the numbers of targets identified in each line. Internal numbers indicate targets common to two or all three lines. (B) Distribution of IRF8-bound sites relative to the transcription start site (TSS) of target genes. (C) Representative IRF8 binding from ChIP-chip in four cell lines. Binding of IRF8 to the TLR4 promoter is shown as an example. Fold change is calculated from relative fold enrichment of IRF8 ChIP signal to input signal. Dashed line shows TSS.
Mapping of probes targeted by IRF8 to the human genome showed that the great majority fell within well-defined peaks located from 1 kb 3′ to 1 kb 5′ from the TSS of involved genes (
An example of the fold enrichment of ChIP to input for each cell line is shown in
(A) ChIP-qPCR validation using primer pairs surrounding the putative binding sites identified by ChIP-chip. For each locus, the fold enrichment comparing IRF8 ChIP DNA to input DNA is represented in the bar graph. The heatmap (lower panel) shows fold enrichment obtained from ChIP-chip. (B) Categorization of IRF8 targets by Gene Ontology (GO). Percents of genes in each category in the whole array or in the set of IRF8 targets are shown. p-values indicate significance of the enrichment for IRF8 targets in each GO category. (C) Motif analysis for IRF8 ChIP hits. Over-represented motifs were identified by TRAWLER and MEME. (D) A Venn diagram of IRF8 targets and interferon-responsive genes. The Interferome DB was used for identifying interferon-responsive genes.
An examination of the genes identified as having IRF8 binding sites by ChIP-chip was performed by Gene Ontology (GO) analysis and revealed significant enrichments for immune response categories including innate and humoral responses, responses to virus as well as antigen processing and presentation (
To identify the characteristics of the cis-regulatory motifs over-represented in the set of IRF8-bound targets, repeat-masked ChIP sequences were queried in TRAWLER
Both IFNα/β and IFNγinitiate transcriptional activation of IFN-stimulated genes (ISGs) by activation of the JAK-STAT signaling pathways
Taken together, the results of our ChIP-chip analyses of human GC-derived lymphoma cell lines identified over 250 target genes with binding sites located primarily at TSSs. The target sites were highly enriched for two distinct binding motifs very similar to canonical ISRE and EICE elements, respectively. A high proportion of the target genes were included in the Interferome of ISGs and were functionally involved in aspects of both innate and acquired immunity including antigen processing and presentation.
Our initial impetus for studying possible contributions of IRF8 to B cell development and function came from analyses of mouse B cell lineage lymphomas showing that levels of IRF8 expression varied significantly at progressive stages of differentiation. Expression was highest in diffuse large B cell lymphoma (DLBCL) of GC origin but was almost totally absent in tumors of mature plasma cells
ChIP-chip analyses identified 3,659 and 2,672 IRF8 binding sites in NFS-201 and NFS-202, respectively, but only 1,290 sites in NFS-205 (
Labeled IRF8 ChIP samples and input samples were applied to Nimblegen MM8 385 k arrays and peak signals were identified by sliding window algorithm with threshold of FDR<0.01. (A) A Venn diagram for IRF8 targets. Numbers in parentheses indicate the number of IRF8 targets identified in each line. Venn diagram shows the number of IRF8 targets that belong to each area. (B) Western blots for IRF8 and PU.1 in the three cell lines. (C) Distribution of IRF8 ChIP hits by chromosomal location relative to transcription start sites (TSS). (D) Distribution of IRF8 binding related to the annotated structure of associated genes (top). The frequencies of IRF8 hits in the sub-structure of intragenic locations (bottom).
The lower number of IRF8 target sites identified in the NFS-205 cell line was also of interest. IRF8 differs from other members of the IRF family in that it can bind DNA only after heterodimerization with other members of the IRF family or with non-IRF transcription factors such as PU.1
Mapping of probes targeted by IRF8 in the three cell lines to the mouse genome paralleled studies of human IRF8 target sites with the majority mapping within 1 kb upstream or downstream of the TSSs of involved genes (
A more detailed characterization of the target sites for their localization to proximal or distal promoters and to intragenic regions is presented in
These results indicated that genes targeted by IRF8 in germinal center cells of both humans and mice are most often characterized by binding sites in proximity to TSSs while also suggesting that the differential distribution between proximal and distal promoter regions is likely to be influenced by the availability of PU.1 as a partner protein.
To identify genes affected at the transcriptional level by alterations in IRF8 expression, we performed gene expression profiling of NFS-202 cells stably transfected with an IRF8-specific or a control siRNA
Total RNAs from NFS-202 cell lines stably expressing siIRF8 and control cell lines were applied to NIAID mouse expression arrays. (A) Significant Analysis of Microarray (SAM) plots for identification of differentially expressed genes in knock-down cell lines. Both up- and down-regulated genes were identified with FDR<0.01. (B) qPCR validation of differentially expressed genes from microarray analysis. Fold change of siIRF8 cell lines vs. control cell lines were plotted against fold change in microarray. Values are in log2 scale. Linear regression analysis was performed (p<0.0001). (C) Functional classification of significant genes in IRF8 knock-down cell lines. Fisher's exact test was performed to identify significantly enriched biological categories using Ingenuity Pathway Analysis (IPA). Log-transformed p-values from Fisher's exact test are shown on the x-axis. (D) GSEA analysis of mRNA expression profiles for IRF8 knock-down cells vs. control cells. Relative expression was rank-ordered by fold change of five replicate IRF8 knock-down samples vs. five replicate control samples. Genes associated with IRF8 binding sites (IRF8 ChIP targets) were strongly correlated with IRF8 expression level. The color bar at the bottom indicates up-regulated (red) and down-regulated (blue) genes. (E) Distribution of PU.1 ChIP hits by chromosomal location relative to transcription start sites (TSS). PU.1 ChIP-chip analyses were done using NFS-201 and NFS-202 cells that express PU.1 and NFS-205 cells that are PU.1-negative. Labeled PU.1 ChIP samples and input samples were applied to Nimblegen MM8 385 k arrays and peak signals were analyzed by sliding window algorithm with threshold of FDR<0.01. (F) Classification of mouse IRF8 targets with altered expression by siIRF8 in relation to PU.1 binding to the gene. A Venn diagram of genes bound by IRF8 from ChIP-chip and genes that were significantly altered in IRF8 knock-down cells as indicated by gene expression microarray (false discovery rate<0.01). Those IRF8 targets with differential expression were classified into two groups based upon the observation of PU.1 binding from ChIP-chip. The top over-represented motifs were identified in two groups of IRF8 ChIP-hits by TRAWLER using mouse 1 kb promoter set as background.
A gene ontology (GO) assessment of genes affected transcriptionally by downregulation of Irf8 revealed enriched gene clusters associated with a variety of cellular processes centered on hematopoietic differentiation as well as cell-mediated and humoral immune responses (
We next applied Gene Set Enrichment Analysis (GSEA) to determine if the expression of genes identified as targets of IRF8 binding by ChIP-chip was altered by suppressing IRF8 expression in NFS-202 cells (
PU.1 is a key transcription factor required for the development of all hematopoietic cells
To examine this possibility, we first used ChIP-chip analyses to characterize PU.1 target sites in NFS-201 and NFS-202 cells with NFS-205 serving as a negative control and identified 1,764 target sites common to both IRF8-expressing cell lines (
By combining ChIP-chip and gene expression microarray studies of the mouse cell lines, we identified 277 genes that were targeted by IRF8 and that were significantly altered in expression in siIRF8 knockdown cells (
A broader picture of the transcriptional landscape governed by IRF8 in mouse B cells is presented in
Activated | Repressed | Not determined | |
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Ifi35, |
Crry, Ddx58, |
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Aif1, Arnt, |
Arts1, Blr1, |
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Gadd45g, Hus1, Parp9, Rad51l1, |
Bard1, |
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Cysltr2, Gem, Gnaz, |
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Akap13, Fgfr1op2, |
Aurkb, |
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Cdca1, Rnf123, Tbrg1, Trp53 | |
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Armcx3, |
Baz2b, Gtf3c5, |
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Cd164, Clec1a, |
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Ehd2, Ide, Katna1, |
Clasp10, Marcks, Mast3, |
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Edem1, |
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Ctrl, Ctso, |
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Cstf2t, |
Cpsf2, |
Ddx21, Ell3, |
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Fbxo17, Fbxo36, Fbxo39, Fbxo43, Parp14, Parp9, |
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Cct6b, |
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Cutc, |
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Cyp4v3, Fars2, Mrpl32 | Cap1, Mrpl3, Mrpl13, Oxsm | |
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Acad8, Plscr1, |
364 IRF8 targets with altered expression by siIRF8 were classified to their functional involvement in GC B cells (only known genes were listed). Genes in
Comparisons of genome-wide transcription factor binding patterns across species indicate that a large proportion of enhancers are species specific with significant divergences between human and mouse
IRF8 targets common in both human and mouse were identified by intersecting human and mouse ChIP-chip analysis. These 51 targets are listed along with data on fold enrichment of IRF8 ChIP vs. input. Data obtained from human cell lines LY1, ODH1 and VAL are shown in the left most heatmap. IRF8 ChIP-chip data from mouse cell lines NFS-201, NFS-202 and NFS-205 are shown in blue. Fold enrichment of PU.1 ChIP vs. input from NFS-201 and NFS-202 are shown in purple. IRF8 expression (Expr) column indicates expression levels of genes identified as activated or repressed by IRF8. Reported responsiveness of genes to interferon type I, II, or III is shown in pale blue in the rightmost map. Numbers in ChIP-chip data are fold enrichment and those in expression array are fold change of control vs. siIRF8 cell lines.
These observations prompted us to validate this network by studying expression levels of MHC class II genes and CIITA in B220 gated FAS+GL7+ GC B cells of IRF8 conventional KO and control mice. First, spleen cells from these mice were analyzed by flow cytometry for the levels of the MHC class II expression on GC B cells. Flow cytometric studies showed that the levels of MHCII expressed by GC cells of IRF8 KO mice were significantly lower than for cells of wt mice (MFI fold change = 1.9,
(A) MHC class II expression in GC B cells in IRF8 KO and WT mice. Representative histogram for MHC class II expression in GC B cells (left panel). Dot plot shows significant difference in MHCII expression between two groups. *, p<0.05 from Mann-Whitney test. (B) MHC class II expression in
The results of this study provide the first comprehensive picture of the transcriptional programs and cellular pathways governed by IRF8 in mature B lineage cells as viewed through the lenses provided by analyses of cell lines of GC origin from humans and mice. Our conclusions derive from a synthesis of data from ChIP-chip analyses of IRF8 occupancy of target sites in both species, microarray-based transcriptional profiling of the mouse cell lines, and ChIP-chip analyses of PU.1 target sites in the mouse cells. The findings indicate that IRF8 is involved in the regulation of a large number of genes of known importance to various aspects of the biology of mature B cells.
As illustrated in
IRF8 targets in B cells were presented in context of signaling pathways in GC.(A) Cross-talk between B cells and T cells and follicular DC in GC. IRF8 targets in B cells are shown together with ligands or products from TFH or FDC cells. (B) Signaling downstream of IL21R, CD40, and BCR. IRF8 targets are shown in red.
Although not illustrated here, there are a large number of cell membrane, cytosolic and endosomal proteins encoded by IRF8 targeted genes that function as sensors of pathogen-associated molecular patterns. It is increasingly well recognized that when engaged, these molecules can exert major influences on B cell activation induced by BCR ligation or signaling through other receptors
The molecular transitions required for the maturation of GC B cells to plasma cells are governed by a relatively small set of transcription factors that lie downstream of signals generated by engagement of the IL21R, CD40 and the BCR (
Our systemic and comprehensive approaches have elucidated the roles played by IRF8 in governing transcriptional network in GC B cells. However, understanding the full nature of IRF8 contributions to B cell biology from the earliest stages of lineage commitment to terminal differentiation will require more detailed investigations of the partnering of IRF8 with other proteins at its target sites. As noted previously, IRF8 can bind DNA only after heterodimerization with other transcription factors. While our studies demonstrated that IRF8 associates with PU.1 at about half of the target sites defined in GC B cells, the full picture of IRF8 bound to these sites may be even more complex as IRF8 has been shown to physically associate with both PU.1 and IRF4 to regulate gene expression through recognition of ISRE and EICE sequence elements
Human lymphoma cell lines of GC origin - LY1, ODH1, and VAL - were kindly provided by Dr. Riccardo Dalla-Favera (Columbia University). LY1 and VALB are GC B cell type DLBCL. ODH1 is a Burkitt lymphoma cell line of type I latency for EBV infection
ChIP was performed according to the manufacturer's protocol (Nimblegen, Reykjavík, Iceland). Briefly, cell lines were cross-linked with formaldehyde and the chromatin extracts were sonicated (Misonix Sonicater 3000). Following immunoprecipitation with anti- IRF8 antibody (sc-6058x, Santa Cruz Biotechnology), DNAs were purified from ChIP samples and input control samples. Purified DNAs were blunt ended, ligated with linkers and amplified by PCR. Amplified ChIP samples and input DNA samples were labeled with Cy3 and Cy5, respectively. Labeled samples were pooled and hybridized onto HG18 385 k two array set for human samples and MM8 385 k two array set for mouse samples. ∼60,000 transcripts and ∼48,000 transcript were represented in the human promoter arrays and mouse promoter arrays, respectively. Arrays were scanned (NimbleGene MS 200). Peaks were detected by searching for four or more probes with signals above the cutoff value, which was a hypothetical maximum, mean+6SD, using a 500 bp sliding window. False discovery rate (FDR) score was calculated with 20 times randomization (GSE30356 in GEO). ChIP targets were functionally classified based on GO (
Genomic sequences were retrieved from the UCSC databases for HG18 and MM8. Over-represented motifs were analyzed using Trawler in EMBL (
ChIP samples were also analyzed by qPCR. Primer designs were based on IRF8 binding sequences from the ChIP-chip data; primer sequences are listed in
Total RNAs prepared from six replicate samples of NFS-202 cell lines stably transfected with IRF8 knockdown siRNAs
The methods used for qPCR were described previously
Western blotting was performed as described previously
Cells were prepared and stained as previously reported
Splenic B cells were purified with DynalBeads (Invitrogen) and cultured at 1×106/mL in 24 well plates with RPMI media containing 10% FBS and 1% penicillin/streptomycin at 37°C. Cells were activated with IFNγ (500 ng/ml) for 2 days. Total RNAs were extracted and transcript levels of MHCII and C2ta were measured by qPCR.
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We thank Dr. Hongsheng Wang, Dr. Alexander Kovalchuk, Dr. Janet Hartley and Dr. Ozato Keiko for many helpful discussions.