OCT2 (Octamer Binding Protein 2): An Essential Role in Germinal Center B Cell Differentiation and the “Achilles Heel” of Derived Lymphomas.

Abstract 362 To identify genes requiered for the proliferation and survival of Diffuse Large B Cell Lymphomas (DLBCL) we conducted an “Achilles Heel” RNA interference screen in cell lines model of ABC (Activated B cell-like) and GCB (Germinal Center B-cell like) DLBCL subtypes. One of the most toxic small hairpins RNAs (shRNAs) in this screen targeted Oct2, encoding a POU domain transcriptional activator. Unlike Oct1, which is constitutively expressed in many cell types, Oct2 is primarily lymphoid restricted. It was identified by virtue of its ability to bind the highly conserved DNA octamer motif (ATGCAAAT) within immunoglobulin (Ig) genes promoters. The B cell specific co-activator OCA-B interacts with the POU domain of the octamer binding proteins enhancing their transactivation potential. Although Oct2 and OCA-B are not essential for Ig transcription, they are required for germinal center (GC) B cell differentiation. To understand the massive apoptotic cell death of DLBCL cells following shRNA Oct2 induction we investigated the genetic pathways controlled by Oct2. We profiled gene expression changes in DLBCL cell lines after knocking down Oct2 and merged this data set with data from genome-wide assessment of Oct2 and OCA-B binding sites, coupling chromatin immunoprecipitation (ChiP) with high-throughput sequencing technologies (ChIP-Seq). ChIP-Seq uncovered an extensive network of Oct2 target genes in DLBCL cells. More than 60% of the Oct2 target genes also showed OCA-B biding. This Oct2/OCA-B overlaping set of targets was enriched for genes selectively expressed in pan-B cells and GC B cells. We found that Oct2/OCA-B lie upstream many of the main transcription factors known to play an essential role in inducing and mantaining the GC stage of B cell development such as BCL6, MTA3, PU.1, IRF8, SpiB and OCA-B, among others. Oct2/OCA-B target these genes both in DLBCL cells and in normal human primary centroblasts. Strikingly, among Oct-2 downstream effectors, BCL6 cDNA was enough to rescue both ABC and GCB DLBCL cells from the Oct2 shRNA lethal effect. The Oct2/OCA-B binding of BCL6 promoter was confirmed in vivo by single locus ChIP in different GCB-DLBCL cell lines as well as in primary centroblasts isolated from human tonsils. Gel shifts experiments showed Oct2 binding to more than one non canonical octamer motif within the BCL6 promoter. Furthermore, computational analysis of the BCL6 promoter region bound by Oct2, showed PU.1 binding sites. Knocked down of PU.1 decreased Oct2 enrichment and viceversa suggesting cooperative Oct2/PU.1 biding to BCL6 pomoter. ChIP-Seq findings opened an entire and exciting new chapter in the Oct2 biology field. Pou transcription factors were suppossed to regulate the activity of octamer containing promoters. Interestingly, Oct2 binds to and control the expression of many GC specific genes that do not harbor a “canonical octamer” motif. Eventhough Oct2 is expressed throughout the different stages of B cell maturation, both mRNA and protein levels are enhanced in centroblasts as compared to pre-GC B cells. We found Oct2 capable of inducing its own expression as well as Oct-1 and OCA-B. This autoregulatory circuit might partially account for the Oct2 predominant role in GC specific genes expression control. By array cGH, high level amplification of Oct2 and OCA-B was found in less than 10% of non Hodgkin lymphoma patients samples. Nonetheless, these lymphoma cells become addicted to the Oct2 controlled network that sustain cell survival and proliferation, wich turns Oct2 into an attractive therapeutic target for non Hodking lymphoma patients. This critical Oct2 DLBCL cells dependency, is an example of “non oncogene addiction” that we recently defined based on our RNA interference screening. Oct-2 controls a network of regulatory relationships that sustain both normal GC B cells and malignant counterparts. In summary, we showed that Oct2 and OCA-B lie upstream of BCL6, one of the critical regulators of germinal center B cell differentiation. This suggests that Oct2-directed therapy should kill the same DLBCLs as BCL6-directed therapy. Furthermore, all GC and Post-GC B cells that were tested requiere Oct-2 for survival, indicating that Oct2-directed therapy might have a broder activity spectrum than the BCL6-directed therapy. The Oct2/OCA-B binding interface would be amenable to attack with potential manageable toxicity, since this interaction is exclusively required in GC B cells. Disclosures: No relevant conflicts of interest to declare.

Role of Defective Oct-2 and OCA-B Expression in Immunoglobulin Production and Kaposi's Sarcoma-Associated Herpesvirus Lytic Reactivation in Primary Effusion Lymphoma

ABSTRACT Primary effusion lymphoma (PEL) is a distinct type of B-cell non-Hodgkin lymphoma characterized by the presence of Kaposi's sarcoma-associated herpesvirus (KSHV/human herpesvirus 8). Despite having a genotype and gene expression signature of highly differentiated B cells, PEL does not usually express surface or cytoplasmic immunoglobulin (Ig). We show the lack of Oct-2 and OCA-B transcription factors to be responsible, at least in part, for this defect in Ig production. Like Ig genes, ORF50, the key regulator of the switch from latency to lytic reactivation, contains an octamer motif within its promoter. We therefore examined the impact of Oct-2 and OCA-B on ORF50 activation. The binding of Oct-1 to the ORF50 promoter has been shown to significantly enhance ORF50 transactivation. We found that Oct-2, on the other hand, inhibited ORF50 expression and consequently lytic reactivation by competing with Oct-1 for the octamer motif in the ORF50 promoter. Our data suggest that Oct-2 downregulation in infected cells would be favorable to KSHV in allowing for efficient viral reactivation.

Transcriptional Regulation of AQP-8, a Caenorhabditis elegans Aquaporin Exclusively Expressed in the Excretory System, by the POU Homeobox Transcription Factor CEH-6

Due to the ever changing environmental conditions in soil, regulation of osmotic homeostasis in the soil-dwelling nematode Caenorhabditis elegans is critical. AQP-8 is a C. elegans aquaporin that is expressed in the excretory cell, a renal equivalent tissue, where the protein participates in maintaining water balance. To better understand the regulation of AQP-8, we undertook a promoter analysis to identify the aqp-8 cis-regulatory elements. Using progressive 5′ deletions of upstream sequence, we have mapped an essential regulatory region to roughly 300 bp upstream of the translational start site of aqp-8. Analysis of this region revealed a sequence corresponding to a known DNA functional element (octamer motif), which interacts with POU homeobox transcription factors. Phylogenetic footprinting showed that this site is perfectly conserved in four nematode species. The octamer site's function was further confirmed by deletion analyses, mutagenesis, functional studies, and electrophoretic mobility shift assays. Of the three POU homeobox proteins encoded in the C. elegans genome, CEH-6 is the only member that is expressed in the excretory cell. We show that expression of AQP-8 is regulated by CEH-6 by performing RNA interference experiments. CEH-6's mammalian ortholog, Brn1, is expressed both in the kidney and the central nervous system and binds to the same octamer consensus binding site to drive gene expression. These parallels in transcriptional control between Brn1 and CEH-6 suggest that C. elegans may well be an appropriate model for determining gene-regulatory networks in the developing vertebrate kidney.

Embryonic Lethality, Decreased Erythropoiesis, and Defective Octamer-Dependent Promoter Activation in Oct-1-Deficient Mice

ABSTRACT Oct-1 is a sequence-specific DNA binding transcription factor that is believed to regulate a large group of tissue-specific and ubiquitous genes. Both Oct-1 and the related but tissue-restricted Oct-2 protein bind to a DNA sequence termed the octamer motif (5′-ATGCAAAT-3′) with equal affinity in vitro. To address the role of Oct-1 in vivo, an Oct-1-deficient mouse strain was generated by gene targeting. Oct-1-deficient embryos died during gestation, frequently appeared anemic, and suffered from a lack of Ter-119-positive erythroid precursor cells. This defect was cell intrinsic. Fibroblasts derived from these embryos displayed a dramatic decrease in Oct-1 DNA binding activity and a lack of octamer-dependent promoter activity in transient transfection assays. Interestingly, several endogenous genes thought to be regulated by Oct-1 showed no change in expression. When crossed to Oct-2 +/− animals, transheterozygotes were recovered at a very low frequency. These findings suggest a critical role for Oct-1 during development and a stringent gene dosage effect with Oct-2 in mediating postnatal survival.

Cellular transcription factors that interact with p6 promoter elements of parvovirus B19

All transcripts of the human parvovirus B19 identified so far are regulated by a single promoter at map unit 6 of the viral genome, the so-called p6 promoter. This promoter is active in a wide variety of different cells. In order to identify cellular transcription factors involved in regulating promoter activity, we performed gel-retardation and supershift assays using the parts of the p6 promoter sequence shown previously to be protected in footprint experiments. Thereby, binding was demonstrated of the Oct-1 protein to an octamer motif within the p6 promoter and of the transcription factor Sp1 to three GC boxes. A specific preferential interaction of the factor Sp3 with one of these boxes was observed, indicating that the ratio Sp1:Sp3 may be involved in the regulation of promoter activity. Consensus sites for the regulatory protein YY1 are located close to the GC boxes and the octamer motif, to which this factor binds efficiently.

Defective octamer-dependent transcription is responsible for silenced immunoglobulin transcription in Reed-Sternberg cells

The absence of immunoglobulin (Ig) expression in B-cell–derived Hodgkin and Reed-Sternberg (HRS) cells of classical Hodgkin disease (cHD) was initially suggested to be caused by crippling mutations in the Ig promoter or coding region. More recent investigations have, however, challenged this concept. This study addressed the role of mutations in the Ig promoter region in HRS cells. Nine cases of cHD and 3 B-cell–derived HD lines were analyzed for mutations in the TATA box and octamer motif of the Ig promoter. Mutations in the octamer motif were found in only 1 of the 9 cases and in 1 of the 3 HD cell lines (L1236). Furthermore, in all cases either a complete lack or strong reduction in the expression of the Oct2 transcription factor and the BOB.1/OBF.1 coactivator were found. The relevance of the rare promoter mutations was investigated by assaying the activity of Ig promoter reporter constructs transfected into the HD cell line L1236, which harbors a mutated octamer motif. These Ig reporter constructs were completely inactive in L1236 cells; however, their activity could be reconstituted by the cotransfection of a BOB.1/OBF.1 expression vector. The additional transfection with an Oct2 expression vector did not further enhance the Ig promoter activity. The conclusions drawn from these results are that crippling mutations in the Ig promoter and coding region are not the sole cause for the lack of Ig expression in HRS cells and that defects in the transcription machinery such as absence of BOB.1/OBF.1 are more important for this phenomenon.

The Gene for the Embryonic Stem Cell Coactivator UTF1 Carries a Regulatory Element Which Selectively Interacts with a Complex Composed of Oct-3/4 and Sox-2

ABSTRACT UTF1 is a transcriptional coactivator which has recently been isolated and found to be expressed mainly in pluripotent embryonic stem (ES) cells (A. Okuda, A. Fukushima, M. Nishimoto, et al., EMBO J. 17:2019–2032, 1998). To gain insight into the regulatory network of gene expression in ES cells, we have characterized the regulatory elements governing UTF1 gene expression. The results indicate that the UTF1 gene is one of the target genes of an embryonic octamer binding transcription factor, Oct-3/4. UTF1 expression is, like the FGF-4 gene, regulated by the synergistic action of Oct-3/4 and another embryonic factor, Sox-2, implying that the requirement for Sox-2 by Oct-3/4 is not limited to the FGF-4 enhancer but is rather a general mechanism of activation for Oct-3/4. Our biochemical analyses, however, also reveal one distinct difference between these two regulatory elements: unlike the FGF-4 enhancer, the UTF1 regulatory element can, by its one-base difference from the canonical octamer-binding sequence, selectively recruit the complex comprising Oct-3/4 and Sox-2 and preclude the binding of the transcriptionally inactive complex containing Oct-1 or Oct-6. Furthermore, our analyses reveal that these properties are dictated by the unique ability of the Oct-3/4 POU-homeodomain that recognizes a variant of the Octamer motif in the UTF1 regulatory element.

Somatic Mutations Within the Untranslated Regions of Rearranged Ig Genes in a Case of Classical Hodgkin’s Disease as a Potential Cause for the Absence of Ig in the Lymphoma Cells

Hodgkin–Reed-Sternberg (H-RS) cells are clonal B cells carrying Ig gene rearrangements. However, in situ hybridization methods failed to demonstrate Ig gene expression in H-RS cells of classical Hodgkin’s disease (HD). Because somatic mutations rendering potentially functional Ig gene rearrangements nonfunctional were detected in some cases of the disease, it was speculated that H-RS cells in classical HD may have lost the ability to express antigen receptor as a rule. Recently, we established a novel cell line (L1236) from H-RS cells of a patient with mixed cellularity subtype of HD. L1236 cells harbor a potentially functional VH1 and a potentially functional Vκ3 gene rearrangement. However, no antibody expression was detected. To show potential reasons for this lack of Ig expression, we analyzed the genomic organization of the Ig genes and their transcription in the primary and cultivated H-RS cells of this patient. The H-RS cells were found to have switched their isotype to IgG4, confirming their mature B-cell nature. By amplifying cDNA from L1236 cells as well as from frozen biopsy material transcripts of the Vκ3 and the VH1 gene rearrangement were detected for both sources of cDNA. However, Northern blot hybridization of L1236 RNA failed to demonstrate VH1 and Vκ3 transcripts, indicating only a low level of transcription. Sequence analysis of the promoter and leader regions of the VH1 gene rearrangement from L1236 cells as well as from lymphoma-affected tissue showed a somatic mutation in the conserved octamer motif of the promoter region. Somatic mutations were also detected within the 3′ splice site of the leader intron and adjacent nucleotides in the rearranged Vκ light chain gene, leading to aberrant splicing. These mutations might prevent the generation of adequate amounts of functional Ig gene transcripts as template for translation into protein. Thus, mutations in H-RS cells that prevent Ig gene expression might also be located outside the coding region of the Ig genes.