SUMMARY Interleukin-21 (IL-21) is a pleiotropic cytokine that induces expression of transcription factor BLIMP1 (encoded by Prdm1), which regulates plasma cell differentiation and T cell homeostasis. We identified an IL-21 response element downstream of Prdm1 that binds the transcription factors STAT3 and IRF4, which are required for optimal Prdm1 expression. Genome-wide ChIP-Seq mapping of STAT3- and IRF4-binding sites showed that most regions with IL-21-induced STAT3 binding also bound IRF4 in vivo and furthermore revealed that the noncanonical TTCnnnTAA GAS motif critical in Prdm1 was broadly used for STAT3 binding. Comparing genome-wide expression array data to binding sites revealed that most IL-21-regulated genes were associated with combined STAT3-IRF4 sites rather than pure STAT3 sites. Correspondingly, ChIP-Seq analysis of Irf4−/− T cells showed greatly diminished STAT3 binding after IL-21 treatment, and Irf4−/− mice showed impaired IL-21-induced Tfh cell differentiation in vivo. These results reveal broad cooperative gene regulation by STAT3 and IRF4.

We have identified two intronic regions of mouse prdm1, the gene encoding B lymphocyte-induced maturation protein-1 (Blimp-1), which confer transcriptional repression in response to Bcl-6. The Bcl-6 response element in intron 5, which is conserved between mice and humans, was studied in detail. It binds Bcl-6 in vitro and was shown by chromatin immunoprecipitation to be occupied by Bcl-6 in vivo. Neither Bcl-6 response element functions as a STAT3-response element, showing that STAT3 does not compete with Bcl-6 at these sites. Bcl-6−/− mice confirm the biological importance of Bcl-6-dependent repression of prdm1. These mice have elevated Ab response, increased Ig-secreting cells, and increased Blimp-1+ cells in spleen following immunization and their splenic B cells show accelerated plasmacytic development in vitro.

Plasma cells are terminally differentiated final effectors of the humoral immune response. Plasma cells that result from antigen activation of B-1 and marginal zone B cells provide the first, rapid response to antigen. Plasma cells that develop after a germinal center reaction provide higher-affinity antibody and often survive many months in the bone marrow. Transcription factors Bcl-6 and Pax5, which are required for germinal center B cells, block plasmacytic differentiation and repress Blimp-1 and XBP-1, respectively. When Bcl-6-dependent repression of Blimp-1 is relieved, Blimp-1 ensures that plasmacytic development is irreversible by repressing BCL-6 and PAX5. In plasma cells, Blimp-1, XBP-1, IRF4, and other regulators cause cessation of cell cycle, decrease signaling from the B cell receptor and communication with T cells, inhibit isotype switching and somatic hypermutation, downregulate CXCR5, and induce copious immunoglobulin synthesis and secretion. Thus, commitment to plasmacytic differentiation involves inhibition of activities associated with earlier B cell developmental stages as well as expression of the plasma cell phenotype.

Various studies have demonstrated a role for E2F proteins in the control of transcription of genes involved in DNA replication, cell cycle progression, and cell fate determination. Although it is clear that the functions of the E2F proteins overlap, there is also evidence for specific roles for individual E2F proteins in the control of apoptosis and cell proliferation. Investigating protein interactions that might provide a mechanistic basis for the specificity of E2F function, we identified the E-box binding factor TFE3 as an E2F3-specific partner. We also show that this interaction is dependent on the marked box domain of E2F3. We provide evidence for a role for TFE3 in the synergistic activation of the p68 subunit gene of DNA polymerase ␣ together with E2F3, again dependent on the E2F3 marked box domain. Chromatin immunoprecipitation assays showed that TFE3 and E2F3 were bound to the p68 promoter in vivo and that the interaction of either E2F3 or TFE3 with the promoter was facilitated by the presence of both proteins. In contrast, neither E2F1 nor E2F2 interacted with the p68 promoter under these conditions. We propose that the physical interaction of TFE3 and E2F3 facilitates transcriptional activation of the p68 gene and provides strong evidence for the specificity of E2F function.The ability of the retinoblastoma (Rb) tumor suppressor protein to regulate cell growth is due, at least in part, to its ability to interact with and regulate the E2F family of transcription factors (8, 34). The E2F proteins have been shown to control the expression of a large number of genes involved in DNA replication, cell cycle progression, and cell fate determination. The E2F family is composed of six distinct gene products that form heterodimeric complexes with partners of the DP family. Sequence analysis reveals three distinct subfamilies of E2F genes: the E2F1, E2F2, and E2F3 genes, the E2F4 and E2F5 genes, and the E2F6 gene. This division also coincides with functional distinctions. The E2F1, E2F2, and E2F3 genes are tightly regulated by cell growth and during the cell cycle, whereas E2F4, E2F5 and E2F6 are constitutively expressed.This cell cycle regulation of E2F1, E2F2, and E2F3 transcription is complemented by mechanisms that tightly regulate the accumulation of the proteins. An N-terminal domain unique to E2F1 to E2F3 is responsible for both ubiquitinmediated degradation of the proteins (29) and targeting by the cyclin A/cdk2 kinase, the latter leading to inhibition of DNA binding capacity (7,22,23,53). The E2F proteins also vary in their role as transcriptional regulatory activities. While E2F1 to E2F3 act as positive regulators of transcription, E2F4 and E2F5 appear to function primarily as transcriptional repressors in concert with Rb family members. E2F6 also appears to function as a transcriptional repressor but in a manner independent of Rb (4,10,47,48).Various experiments have suggested distinct functional roles for the activating E2F proteins E2F1, E2F2, and E2F3. The E2F3 protein appears to be particularly i...