Transcription Factor Pip Can Enhance DNA Binding by E47, Leading to Transcriptional Synergy Involving Multiple Protein Domains

Abstract

The transcription factors E2A (E12/E47) and Pip are both required for normal B-cell development. Each protein binds to regulatory sequences within various immunoglobulin enhancer elements. Activity of E2A proteins can be regulated by interactions with other proteins which influence their DNA binding or activation potential. Similarly, Pip function can be influenced by interaction with the protein PU.1, which can recruit Pip to bind to DNA. We show here that a previously unidentified Pip binding site resides adjacent to the E2A binding site within the immunoglobulin 3 enhancer. Both of these binding sites are crucial for high-level enhancer activity. We found that E47 and Pip can functionally interact to generate a very potent 100-fold transcriptional synergy. Through a series of mutagenesis experiments, we identified the Pip sequences necessary for transcriptional activation and for synergy with E47. Two synergy domains (residues 140 to 207 and 300 to 420) in addition to the Pip DNA binding domain (residues 1 to 134) are required for maximal synergy with E47. We also identified a Pip domain (residues 207 to 300) that appears to mask Pip transactivation potential. Part of the synergy mechanism between E47 and Pip appears to involve the ability of Pip to increase DNA binding by E47, perhaps by inducing a conformational change in the E47 protein. E47 may also induce a conformational change in Pip which unmasks sequences important for transcriptional activity. Based upon our results, we propose a model for E47-Pip transcriptional synergy.

Interferon regulatory factor 4 differentially regulates the production of Th2 cytokines in naïve vs. effector/memory CD4+T cells

“…IRF-4 contains an amino-terminal DNA-binding domain and a carboxyl-terminal IRF association domain that can interact with a variety of proteins, including IRFs. IRF-4 interacts with Ets family member PU.1, transcription factor E47, Stat6, Bcl-6 or NFATc2 to synergistically enhance transcriptional activity of a variety of genes including the Ig light chain gene in B cells and IL-4 in T cells (14,19,28,29). However, IRF-4 has been shown to repress gene activation induced by other IRFs and does not appear to require association with other proteins (30,31).…”

“…These results are seemingly paradoxical but are consistent with the results of IRF-4 knockdown experiments using siRNA in naïve vs. effector/memory type CD4 ϩ T cells; the inhibition of IRF-4 expression in naïve CD4 ϩ T cells conferred the ability to produce IL-4 on naïve CD4 ϩ T cells, whereas inhibition of IRF-4 expression in effector/memory CD4 ϩ T cells down-regulated IL-4 expression. Studies on the function of IRF-4 indicated that it could function as either a transcriptional activator or repressor depending on the context of the DNA-binding sequences and/or protein-interacting partners (14,19,(28)(29)(30)(31). IRF-4 contains an amino-terminal DNA-binding domain and a carboxyl-terminal IRF association domain that can interact with a variety of proteins, including IRFs.…”

B-cell receptor activation inhibits AID expression through calmodulin inhibition of E-proteins

“…5D), because IRF-8 has recently been reported to participate in the activation of AID gene expression (15). It is also interesting that anti-IgM reduced the expression of IRF-4 mRNA, because IRF-4 has been shown to synergize with the E2A protein E47 at another promoter, the I promoter activating germ line I transcription, which is required for isotype class switching (28,29). In addition, IRF4-deficient B cells have been shown to display impaired expression of AID and lack class-switch recombination (30).…”

IFN-Stimulated Gene 15 Is Synergistically Activated Through Interactions Between the Myelocyte/Lymphocyte-Specific Transcription Factors, PU.1, IFN Regulatory Factor-8/IFN Consensus Sequence Binding Protein, and IFN Regulatory Factor-4: Characterization of a New Subtype of IFN-Stimulated Response Element

“…However, IRF-8\ICSBP can associate with either IRF-1 or IRF-2, forming heterocomplexes that bind efficiently to the DNA (3,4). Further, both IRF-4 and IRF-8\ICSBP also associate with non-IRF members such as PU.1 or E47 on a DNA composite element, half of which is an IRF binding site and half of which is the binding site of the interacting partner (5,6). These protein-protein interactions are mediated through a conserved domain termed the IRF association domain (IAD) (7).…”