Assembly of a mixed interaction network specific to human B cells.Identification and validation of master regulators of germinal center reaction.MYB and FOXM1 are synergistic master regulators of proliferation in germinal center B cells and control a new protein complex involving replication and mitotic-related genes.
We have assembled an interaction network specific to human B cells (the human B-cell interactome or HBCI), containing protein–DNA and protein–protein interactions using an evidence integration approach. The integration of different interaction layers in one network allowed us to elucidate master regulator (MR) genes controlling specific cellular processes as well as transcriptional regulation of proteins in complexes whose availability must be regulated in context-dependent manner. The latter is a poorly understood process, as transcriptional networks and protein–protein interaction networks are usually studied in isolation. We have developed a new algorithm called master regulator inference analysis (MARINa) for discovering MRs of specific phenotypes and applied it to the HBCI to infer MRs of germinal center (GC) formation. GCs are structures where antigen-stimulated B cells highly proliferate, undergo somatic hypermutation of immunoglobulin genes, and are selected based on the production of high-affinity antibodies. GC B cells (centroblasts) derive from naive B cells, from which they differ for the activation of genetic programs controlling cell proliferation, DNA metabolism, and pro-apoptotic programs and for the repression of anti-apoptotic, cell-cycle arrest, DNA repair, and signal transduction programs from cytokines and chemokines. MARINa recovered known MRs of GC B cells and also revealed a new transcription factor module controlling their proliferation. In particular, we identified MYB and FOXM1 as being key MRs of GC B cells. Indeed, 80% of the genes jointly regulated by these transcription factors are activated in the GC, including those encoding proteins in a predicted complex regulating DNA pre-replication, replication, and mitosis. We first tested whether MYB and FOXM1 may regulate each other as predicted in the HBCI, and show that MYB is a transcriptional activator of FOXM1, suggesting that they form a feed-forward loop, involved in the synergistic activation of a large subset of GC-specific genes. We then validated that common MYB/FOXM1 targets and other predicted MRs were affected by the silencing of either TF, using gene expression profiling. Furthermore, we showed that downregulated targets (AURKA, BUBR1, CCNB2, FANCI, MCM3, and PTTG1) and MRs (NFYB, E2F1, and E2F5) after MYB or FOXM1 silencing are indeed directly bound by them in their promoter region. Silencing of FOXM1 and MYB showed a decrease in proliferating cells and an increase in apoptotic cells, indicating that MYB and FOXM1 are necessary for viability and rapid proliferation of GC B cells. To gain more insight into the control of GC-proliferation phenotype by MYB and FOXM1, we further examined specific targets involved in the formation of a predicted protein complex. Approximately half of MYB/FOXM1 targets cluster within a complex, including new interactions between pre-replication and mitotic proteins. We experimentally validate that two mitotic kinases in the inferred complex, BUBR1 and AURKA, physically interact with MCM3, all of them being confirmed to be direct targets of FOXM1 and MYB. In summary, these results document that coordinated analysis of both transcriptional and post-translational interactions in the HBCI can identify synergistic MRs of human phenotypes, as well as provide insight on the functional regulatory role of these proteins. These results indicate that the HBCI analysis can be used for the identification of determinants of major human cell phenotypes and provides a paradigm of general applicability to normal and pathologic tissues.
Assembly of a transcriptional and post-translational molecular interaction network in B cells, the human B-cell interactome (HBCI), reveals a hierarchical, transcriptional control module, where MYB and FOXM1 act as synergistic master regulators of proliferation in the germinal center (GC). Eighty percent of genes jointly regulated by these transcription factors are activated in the GC, including those encoding proteins in a complex regulating DNA pre-replication, replication, and mitosis. These results indicate that the HBCI analysis can be used for the identification of determinants of major human cell phenotypes and provides a paradigm of general applicability to normal and pathologic tissues.