Michelle Maurin

PRDM1/Blimp-1 Controls Effector Cytokine Production in Human NK Cells

NK cells are major effectors of the innate immune response through cytolysis and bridge to the adaptive immune response through cytokine release. The mediators of activation are well studied; however, little is known about the mechanisms that restrain activation. In this report, we demonstrate that the transcriptional repressor PRDM1 (also known as Blimp-1 or PRDI-BF1) is a critical negative regulator of NK function. Three distinct PRDM1 isoforms are selectively induced in the CD56dim NK population in response to activation. PRDM1 coordinately suppresses the release of IFN-γ, TNF-α, and TNF-β through direct binding to multiple conserved regulatory regions. Ablation of PRDM1 expression leads to enhanced production of IFN-γ and TNF-α but does not alter cytotoxicity, whereas overexpression blocks cytokine production. PRDM1 response elements are defined at the IFNG and TNF loci. Collectively, these data demonstrate a key role for PRDM1 in the negative regulation of NK activation and position PRDM1 as a common regulator of the adaptive and innate immune response.

PRDM1 Is Required for Mantle Cell Lymphoma Response to Bortezomib

Mantle cell lymphoma (MCL) is an aggressive form of B-cell lymphoma with a poor disease-free survival rate. The proteasome inhibitor bortezomib is approved for the treatment of relapsed and refractory MCL and has efficacy in about 30% of patients. However, the precise mechanism of action of bortezomib is not well understood. This report establishes a requirement for the transcription repressor PR domain zinc finger protein 1 (PRDM1, Blimp1) in the response to bortezomib. Bortezomib rapidly induces transcription of PRDM1 as part of the apoptotic response in both cell lines and primary MCL tumor cells. Knockdown of PRDM1 blocks activation of NOXA and inhibits apoptosis, whereas ectopic expression of PRDM1 alone leads to apoptosis in MCL. Two novel direct targets of PRDM1 were identified in MCL cells: MKI67 (Ki67) and proliferating cell nuclear antigen (PCNA). Both MKI67 and PCNA are required for proliferation and survival. Chromatin immunoprecipitation and knockdown studies reveal that specific repression of MKI67 and PCNA is mediated by PRDM1 in response to bortezomib. Furthermore, promoter studies and mutation/deletion analysis show that PRDM1 functions through specific sites in the PCNA proximal promoter and an MKI67 distal upstream repression domain. Together, these findings establish PRDM1 as a key mediator of bortezomib activity in MCL. Mol Cancer Res; 8(6); 907–18. ©2010 AACR.

PU.1 regulates positive regulatory domain I-binding factor 1/Blimp-1 transcription in lymphoma cells.

The human positive regulatory domain I-binding factor 1 (PRDI-BF1) and its murine homolog Blimp-1 promote differentiation of mature B cells into Ab-secreting plasma cells. In contrast, ectopic expression of PRDI-BF1 in lymphoma cells can lead to inhibition of proliferation or apoptosis. However, little is currently known about the regulation of PRDM1, the gene encoding PRDI-BF1. This report establishes that in lymphoma cells stimulation through the BCR rapidly induces endogenous PRDM1 at the level of transcription with minor changes in mRNA stability. The induced PRDM1-encoded protein localizes to its target genes in vivo and suppresses their expression. In vivo genomic footprinting of the PRDM1 promoter in unstimulated lymphoma and myeloma cells reveals multiple common in vivo occupied elements throughout the promoter. Further functional and structural analysis of the promoter reveals that the promoter is preloaded and poised for activation in the B cell lines. The transcription factor PU.1 is shown to be required for the BCR-induced expression of PRDM1 in lymphoma cells and in PU.1-positive myeloma cells. Activation of PRDM1 is associated with loss of the corepressor transducin-like enhancer of split 4 from the PU.1 complex. These findings indicate that PRDM1 is poised for activation in lymphoma cells and therefore may be a potential therapeutic target to inhibit lymphoma cell proliferation and survival.

PRDM1/Blimp1 downregulates expression of germinal center genes LMO2 and HGAL

Human germinal center‐associated lymphoma (HGAL) and LIM domain only‐2 (LMO2) are proteins highly expressed in germinal center (GC) B lymphocytes. HGAL and LMO2 are also expressed in GC‐derived lymphomas and distinguish biologically distinct subgroups of diffuse large B‐cell lymphomas (DLBCL) associated with improved survival. However, little is known about their regulation. PRDM1/Blimp1 is a master regulator of terminal B cell differentiation and may also function as a tumor suppressor in the pathogenesis of DLBCL, where it is frequently inactivated by mutations and deletions. We now demonstrate that both HGAL and LMO2 are directly regulated by the transcription repressor PRDM1. In vivo studies demonstrate that PRDM1 directly binds to the recognition sites within the upstream promoters of both HGAL and LMO2. PRDM1 binding suppresses endogenous protein and mRNA levels of HGAL and LMO2. In addition, promoter analysis reveals that site‐specific binding of PRDM1 to the promoters is capable of repressing transcriptional activity. This inhibitory effect of PRDM1 suggests that it has a key role in the loss of HGAL and LMO2 expression upon differentiation of GC B cells to plasma cells and may also contribute to absence of HGAL and LMO2 expression in post‐GC lymphoid tumors.

Data supporting the functional role of Eleven-nineteen Lysine-rich Leukemia 3 (ELL3) in B cell lymphoma cell line cells

The data presented here are related to the research article entitled “Selective expression of the transcription elongation factor ELL3 in B cells prior to ELL2 drives proliferation and survival” (Alexander et al., 2017) [1]. The cited research article characterizes Eleven-nineteen Lysine-rich Leukemia 3 (ELL3) expression in the B cell compartment and functional dependence in B lymphoma cell lines. This data report describes the mRNA expression pattern in a panel of cell lines representing the B cell compartment, supplementing the protein expression data presented in the associated research report. In addition, a reanalysis is presented of publicly available mRNA expression data from primary murine B cells to reveal dynamic regulation of the ELL family members post LPS stimulation (Barwick et al., 2016) [2]. The effect of ELL3 depletion on cell morphology, latent Epstein Barr Virus (EBV) lytic replication and differentiation markers in a Burkitts lymphoma (BL) cell line cells are presented.

Abstract 2331: Transcriptional repressor PRDM1/Blimp-1 directly regulates transcriptional elongation factor ELL3 during terminal B-cell differentiation

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA As the master regulator of B-cell differentiation, PRDM1 is well known for its ability to extinguish a network of transcription factors essential in maintaining the B-cell phenotype. In addition PRDM1 is a tumor suppressor in Diffuse Large B-Cell Lymphoma (DLBCL). However its molecular targets remain to be clearly elucidated. For that purpose we have identified novel targets of PRDM1 by chromatin immunoprecipitation (ChIP) and deep sequencing. One of the strongest associations with PRDM1 was observed at the Eleven-Nineteen Lysine-rich Leukemia (ELL) 3 promoter. The ELL family of transcriptional elongation factors (ELL, ELL2, and ELL3) is reported to increase the catalytic rate of RNA polymerase (pol) II transcription through assembly of the Super Elongation Complex (SEC), although their role in activated B-cells is unknown. Two potential consensus PRDM1 binding sites were mapped within the ELL3 proximal promoter and endogenous PRDM1 binding was confirmed by direct ChIP-qPCR analysis in multiple cell lines. The cloned ELL3 promoter was highly active in B-cell lines and co-transfection with PRDM1 significantly repressed activity. Mutation of both PRDM1 binding sites eliminated repression, while mutation of either individual site resulted in a partial loss of repression. To establish the expression pattern of ELL family members in normal human B-cells, primary naive B-cells were isolated by negative selection and either mildly activated with IL-2 and IL-4 or differentiated in the presence of IL-2, IL-21, anti-IgM, and CD40 cross-linking antibody. Naive B-cells did not express detectable protein levels of any ELL family members. Mild activation induced the expression of ELL and ELL3 while in contrast differentiation selectively suppressed ELL3 while inducing ELL2 and PRDM1. Expression of ELL, ELL2, and ELL3 was further profiled in a panel of B-cell lymphomas and multiple myeloma cell lines. Cell lines representing mature activated B-cells (Burkitts, DLBCL) displayed robust expression of ELL3 while lines of a pre B-cell, naive B-cell or differentiated plasma cell did not express ELL3. ELL2 expression was observed only in the plasma cell lines, consistent with previous reports. ELL was ubiquitously expressed at low levels. shRNA knockdown of ELL3 in ELL3 expressing B-cell lymphomas reveals that ELL3 is required for cell proliferation. Together these results establish ELL3 as an activated B-cell restricted elongation factor and suggests that a switch from ELL3 to ELL2 occurs during terminal differentiation. Dependence on ELL3 for proliferation of lymphoma lines suggests it may be a valid therapeutic target in ELL3 high expressing tumors and is consistent with suppression by the tumor suppressor PRDM1. Citation Format: Lou-Ella M.M. Alexander, January M. Watters, Michelle Maurin, Kenneth L. Wright. Transcriptional repressor PRDM1/Blimp-1 directly regulates transcriptional elongation factor ELL3 during terminal B-cell differentiation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2331. doi:10.1158/1538-7445.AM2014-2331