Analysis of interleukin-21-induced Prdm1 gene regulation reveals functional cooperation of STAT3 and IRF4 transcription factors.

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.

Regulatory events in early and late B-cell differentiation.

We are studying transcriptional control of critical developmental decision points in B lymphocytes. Commitment to the B-lymphocyte lineage is dependent on the transcriptional regulator Pax5 and committed B lymphocytes represent the first developmental stage when V(H)-to-DJ recombination occurs in the immunoglobulin (Ig) heavy chain locus. We summarize our recent studies showing that methylation of histone H3 lysine 9, a heterochromatic chromatin modification, is present in the Ig V(H) region in hematopoietic progenitors and in non-B lineage hematopoietic cells. Pax5 is both necessary and sufficient to remove this heterochromatic mark in B cells. Using genetically altered mice, we have shown that terminal differentiation of B cells to memory and Ig-secreting plasma cells depends on the transcriptional repressor Blimp-1. Recent studies demonstrating a requirement for Blimp-1 in the formation of pre-plasma memory B cells, Ig-secreting plasma cells as well as preliminary data suggesting a requirement for Blimp-1 in the maintenance of long-lived plasma cells are summarized. We also summarize our recent studies on the regulation of Blimp-1, showing direct repression by Bcl-6 and providing evidence for activation by NF-kappaB following toll-like receptor signaling.

Direct repression of prdm1 by Bcl-6 inhibits plasmacytic differentiation.

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.

ICSBP/IRF-8 inhibits mitogenic activity of p210 Bcr/Abl in differentiating myeloid progenitor cells.

Interferon consensus sequence binding protein/interferon regulatory factor 8 (ICSBP/IRF-8) is a transcription factor that controls myeloid cell development. ICSBP-/- mice develop a chronic myelogenous leukemia (CML)-like syndrome. Several observations on patients and mouse models have implicated ICSBP in the pathogenesis of CML. In this paper, we investigated whether ICSBP modulates the growth-promoting activity of Bcr/Abl, the causal oncoprotein for CML. When transformed with p210 Bcr/Abl, ICSBP-/- myeloid progenitor cells lost growth factor dependence and grew in the absence of granulocyte-macrophage colony-stimulating factor. When ICSBP was ectopically expressed, Bcr/Abl-transformed cells underwent complete growth arrest and differentiated into mature, functional macrophages without inhibiting the kinase activity of Bcr/Abl. Providing a mechanistic basis for the growth arrest, ICSBP markedly repressed c-Myc messenger RNA (mRNA)-expression, a downstream target of Bcr/Abl. A further analysis with the ICSBP/estrogen receptor chimera showed that ICSBP repression of c-Myc is indirect and is mediated by another gene(s). We identified Blimp-1 and METS/PE1, potent c-Myc repressors, as direct targets of ICSBP activated in these cells. Consistent with this, ectopic Blimp-1 repressed c-Myc expression and inhibited cell growth. These results indicate that ICSBP inhibits growth of Bcr/Abl-transformed myeloid progenitor cells by activating several genes that interfere with the c-Myc pathway.

Transcriptional regulatory cascades controlling plasma cell differentiation.

Plasma cells are the terminally differentiated effector cells of the B lymphocyte lineage. Recently, studies using genetically altered mice and analyses of global gene expression programs have significantly expanded our understanding of the molecular mechanisms regulating plasmacytic differentiation. Specific molecular components of a multistep cascade of transcriptional regulators have been identified. Furthermore, two transcriptional regulators, X box binding protein-1 (XBP-1) and B lymphocyte induced maturation protein-1 (Blimp-1), have been shown to be necessary for plasmacytic differentiation. In addition to providing a mechanistic basis for the induction of genes necessary for immunoglobulin secretion, cessation of cell cycle and other phenotypic changes characteristic of terminally differentiated plasma cells, these studies have led to the important concept that plasmacytic differentiation involves repression of regulators, such as Bcl-6 and Pax5, that are necessary to maintain the earlier developmental phenotype of activated, germinal center B cells. This review describes our current understanding of the transcriptional cascades regulating terminal differentiation of B cells.

Identification of E-box factor TFE3 as a functional partner for the E2F3 transcription factor.

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 alpha 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.

Regulatory mechanisms that determine the development and function of plasma cells.

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.