Chromosomal translocations juxtaposing the MYC protooncogene with regulatory sequences of immunoglobulin (Ig) H chain or kappa (Igκ) or lambda (Igλ) L chain genes and effecting deregulated expression of MYC are the hallmarks of human Burkitt lymphoma (BL). Here we report that lymphomas with striking similarities to BL develop in mice bearing a mutated human MYC gene controlled by a reconstructed Igλ locus encompassing all the elements required for establishment of locus control in vitro. Diffusely infiltrating lymphomas with a typical starry sky appearance occurred in multiple founders and an established line, indicating independence from positional effects. Monoclonal IgM+CD5−CD23− tumors developed from an initially polyclonal population of B cells. These results demonstrate that the phenotype of B lineage lymphomas induced by MYC dysregulation is highly dependent on cooperativity among the regulatory elements that govern expression of the protooncogene and provide a new system for studying the pathogenesis of BL.

Numerous genes contain promoter elements that are nuclease hypersensitive. These elements frequently possess polypurine/polypyrimidine stretches and are usually associated with altered chromatin structure. We have previously isolated a clone that binds a class of CT-rich promoter elements. We have further characterized this clone, termed the nuclease-sensitive element protein-1, or NSEP-1. NSEP-1 binds both duplex CT elements and the CT-rich strand of these elements in a 'generic' sequence specific manner and has overlapping but distinct single-and double-strand DNA binding domains. The minimal peptide region sufficient for both duplex and single-strand DNA binding includes two regions rich in basic amino acids flanking an RNP-CS-1 like octapeptide motif. Deletion analysis shows that the single-strand DNA binding activity is mediated by the RNP-CS-1 like octapeptide motif and is the key peptide region necessary for single-strand binding. NSEP-1's affinity for CT rich promoter elements with strand asymmetry in addition to its double- and single-strand DNA binding properties suggests that it may be a member of a class of DNA binding proteins that modulate gene expression by their ability to recognize DNA with unusual secondary structure.

We used gene targeting in mice to insert a His 6 -tagged mouse c-Myc cDNA, MycHis , head to head into the mouse immunoglobulin heavy-chain locus, Igh, just 5V of the intronic enhancer, EA. The insertion of Myc His mimicked both the human t(8;14)(q24;q32) translocation that results in the activation of MYC in human endemic Burkitt lymphomas and the homologous mouse T(12;15) translocation that deregulates Myc in certain mouse plasmacytomas. Beginning at the age of 6 months, MycHis transgenic mice developed Bcell and plasma neoplasms, such as IgM + lymphoblastic B-cell lymphomas, Bcl-6 + diffuse large B-cell lymphomas, and CD138 + plasmacytomas, with an overall incidence of 68% by 21 months. Molecular studies of lymphoblastic B-cell lymphoma, the most prevalent neoplasm (50% of all tumors), showed that the lymphomas were clonal, overexpressed Myc His , and exhibited the P2 to P1 promoter shift in Myc expression, a hallmark of MYC/Myc deregulation in human endemic Burkitt lymphoma and mouse plasmacytoma. Only 1 (6.3%) of 16 lymphoblastic B-cell lymphomas contained a BL-typical point mutation in the amino-terminal transactivation domain of Myc His , suggesting that most of these tumors are derived from naive, pregerminal center B cells. Twelve (46%) of 26 lymphoblastic B-cell lymphomas exhibited changes in the p19ArfMdm2-p53 tumor suppressor axis, an important pathway for Myc-dependent apoptosis. We conclude that Myc His insertion into Igh predictably induces B-cell and plasma-cell tumors in mice, providing a valuable mouse model for understanding the transformation-inducing consequences of the MYC/Mycactivating endemic Burkitt lymphoma t(8;14)/plasmacytoma T(12;15) translocation. (Cancer Res 2005; 65(4): 1306-15)

Interferon (IFN) consensus sequence-binding protein/IFN regulatory factor 8 (IRF8) is a transcription factor that regulates the differentiation and function of macrophages, granulocytes, and dendritic cells through activation or repression of target genes. Although IRF8 is also expressed in lymphocytes, its roles in B cell and T cell maturation or function are ill defined, and few transcriptional targets are known. Gene expression profiling of human tonsillar B cells and mouse B cell lymphomas showed that IRF8 transcripts were expressed at highest levels in centroblasts, either from secondary lymphoid tissue or transformed cells. In addition, staining for IRF8 was most intense in tonsillar germinal center (GC) dark-zone centroblasts. To discover B cell genes regulated by IRF8, we transfected purified primary tonsillar B cells with enhanced green fluorescent protein–tagged IRF8, generated small interfering RNA knockdowns of IRF8 expression in a mouse B cell lymphoma cell line, and examined the effects of a null mutation of IRF8 on B cells. Each approach identified activation-induced cytidine deaminase (AICDA) and BCL6 as targets of transcriptional activation. Chromatin immunoprecipitation studies demonstrated in vivo occupancy of 5′ sequences of both genes by IRF8 protein. These results suggest previously unappreciated roles for IRF8 in the transcriptional regulation of B cell GC reactions that include direct regulation of AICDA and BCL6.

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The striking similarity between the treatments that induce SOS functions and those that result in stable DNA replication (continuous DNA replication in the absence of protein synthesis) prompted us to examine the possibility of stable DNA replication being a recA+ lexA+-dependent SOS function. In addition to the treatments previously reported, ultraviolet (UV) irradiation or treatment with mitomycin C was also found to induce stable DNA replication. The thermal treatment of tif-1 strains did not result in detectable levels of stable DNA replication, but nalidixic acid readily induced the activity in these strains. The induction of stable DNA replication with malidixic acid was severely suppressed in tif-1 lexA mutant strains. The inhibitory activity of lexA3 was negated by the presence of the spr-51 mutation, an intragenic suppressor of lexA3. Induced stable DNA replication was found to be considerably more resistant to UV irradiation than normal replication both in a uvrA6 strain and a uvr+ strain. The UV-resistant replication occurred mostly in the semiconservative manner. The possible roles of stable DNA replication in repair of damaged DNA are discussed.

Previously we demonstrated that murine retroviral Gag proteins associate with a cellular motor protein, KIF-4. Using the yeast two-hybrid assay, we also found an association of KIF-4 with Gag proteins of Mason-Pfizer monkey virus (MPMV), simian immunodeficiency virus (SIV), and human immunodeficiency virus type 1 (HIV-1). Studies performed with mammalian cell systems confirmed that the HIV-1 Gag protein associates with KIF-4. Soluble cytoplasmic proteins from cells infected with recombinant vaccinia virus expressing the entire Gag-Pol precursor protein of HIV-1 or transfected with HIV-1 molecular clone pNL4-3 were fractionated by sucrose gradient centrifugation and further separated by size-exclusion and anion-exchange chromatographies. KIF-4 and HIV-1 Gag cofractionated in both chromatographic separations. Immunoprecipitation assays have also verified the KIF-4–Gag association. KIF-4 binds mainly to the Gag precursor (Pr55 Gag) and a matrix-capsid processing intermediate (Pr42) but not to other processed Gag products. The binding of Gag is mediated by a domain of KIF-4 proximal to the C terminus. These results, and our previous studies, raise the possibility that KIF-4 may play an important role in retrovirus Gag protein transport.

Germline mutations in Fas and Fasl induce nonmalignant T cell hyperplasia and systemic autoimmunity and also greatly increase the risk of B cell neoplasms. B lymphomas occurring in Fasl mutant (gld) mice usually are immunoglobulin (Ig) isotype switched, secrete Ig, and are plasmacytoid in appearance but lack Myc translocations characteristic of other plasma cell (PC) neoplasms. Here, we explore the relationship between B cell autoreactivity and transformation and use gene expression profiling to further classify gld plasmacytoid lymphomas (PLs) and to identify genes of potential importance in transformation. We found that the majority of PLs derive from antigen-experienced autoreactive B cells producing antinuclear antibody or rheumatoid factor and exhibit the skewed Ig V gene repertoire and Ig gene rearrangement patterns associated with these specificities. Gene expression profiling revealed that both primary and transplanted PLs share a transcriptional profile that places them at an early stage in PC differentiation and distinguishes them from other B cell neoplasms. In addition, genes were identified whose altered expression might be relevant in lymphomagenesis. Our findings provide a strong case for targeted transformation of autoreactive B cells in gld mice and establish a valuable model for understanding the relationship between systemic autoimmunity and B cell neoplasia.