Lack of somatic hypermutation of IG V(H) genes in lymphoid malignancies with t(2;14)(p13;q32) translocation involving the BCL11A gene.

The t(2;14)(p13;q32.3) involving the BCL11A and IGH genes is a rare but recurrent chromosomal aberration in B-cell malignancies. Hitherto, juxtaposition of BCL11A and IGH has only been described in B-cell chronic lymphocytic leukemia (B-CLL) and immunocytoma. As subgroups of B-CLL can be distinguished by the pattern of somatic mutation of immunoglobulin variable (V) genes we investigated four lymphomas with IGH/BCL11A involvement for IGH hypermutation. Clonal V(H) gene rearrangements were amplified; in all four cases, sequencing of the amplificates revealed the rearranged V(H) genes to lack somatic mutations. These results suggest that t(2;14)(p13;q32.3) is associated with a subset of B-CLL/immunocytoma characterized by non-mutated IG genes deriving from pre-germinal center B cells. As the translocations in both informative cases are targeted to the switch regions of the IGG2 gene, which is mainly used in T cell-independent immune responses, these translocations presumably occurred in activated B cells in the course of T cell-independent immune responses outside the germinal center.

Lymphoid apoptosis and myeloid hyperplasia in CCAAT displacement protein mutant mice.

CCAAT displacement protein (cux/CDP) is an atypical homeodomain protein that represses expression of several developmentally regulated lymphoid and myeloid genes in vitro, including gp91-phox, immunoglobulin heavy chain, the T-cell receptor beta and gamma chains, and CD8. To determine how this activity affects cell development in vivo, a hypomorphic allele of cux/CDP was created by gene targeting. Homozygous mutant mice (cux/CDP(Delta HD/Delta HD)) demonstrated a partial neonatal lethality phenotype. Surviving animals suffered from a wasting disease, which usually resulted in death between 2 and 3 weeks of age. Analysis of T lymphopoiesis demonstrated that cux/CDP(Delta HD/Delta HD) mice had dramatically reduced thymic cellularity due to enhanced apoptosis, with a preferential loss of CD4(+)CD8(+) thymocytes. Ectopic CD25 expression was also observed in maturing thymocytes. B lymphopoiesis was also perturbed, with a 2- to 3-fold reduction in total bone marrow B-lineage cells and a preferential loss of cells in transition from pro-B/pre-BI to pre-BII stages due to enhanced apoptosis. These lymphoid abnormalities were independent of effects related to antigen receptor rearrangement. In contrast to the lymphoid demise, cux/CDP(Delta HD/Delta HD) mice demonstrated myeloid hyperplasia. Bone marrow reconstitution experiments identified that many of the hematopoietic defects were linked to microenvironmental effects, suggesting that underexpression of survival factors or overexpression of death-inducing factors accounted for the phenotypes observed. Tumor necrosis factor (TNF) levels were elevated in several tissues, especially thymus, suggesting that TNF may be a target gene for cux/CDP-mediated repression. These data suggest that cux/CDP regulates normal hematopoiesis, in part, by modulating the levels of survival and/or apoptosis factors expressed by the microenvironment.

The BCL11 gene family: involvement of BCL11A in lymphoid malignancies.

Many malignancies of mature B cells are characterized by chromosomal translocations involving the immunoglobulin heavy chain (IGH) locus on chromosome 14q32.3 and result in deregulated expression of the translocated oncogene. t(2;14)(p13;q32.3) is a rare event in B-cell malignancies. In contrast, gains and amplifications of the same region of chromosome 2p13 have been reported in 20% of extranodal B-cell non-Hodgkin lymphomas (B-NHL), in follicular and mediastinal B-NHL, and in Hodgkin disease (HD). It has been suggested that REL, an NF-kappaB gene family member, mapping within the amplified region, is the pathologic target. However, by molecular cloning of t(2;14)(p13;q32.3) from 3 cases of aggressive B-cell chronic lymphocytic leukemia (CLL)/immunocytoma, this study has shown clustered breakpoints on chromosome 2p13 immediately upstream of a CpG island located about 300 kb telomeric of REL. This CpG island was associated with a Krüppel zinc finger gene (BCL11A), which is normally expressed at high levels only in fetal brain and in germinal center B-cells. There were 3 major RNA isoforms of BCL11A, differing in the number of carboxy-terminal zinc fingers. All 3 RNA isoforms were deregulated as a consequence of t(2;14)(p13;q32.3). BCL11A was highly conserved, being 95% identical to mouse, chicken, and Xenopus homologues. BCL11A was also highly homologous to another gene (BCL11B) on chromosome 14q32.1. BCL11A coamplified with REL in B-NHL cases and HD lymphoma cell lines with gains and amplifications of 2p13, suggesting that BCL11A may be involved in lymphoid malignancies through either chromosomal translocation or amplification.

Transcriptional activation by a matrix associating region-binding protein. contextual requirements for the function of bright.

Bright (B cell regulator of IgH transcription) is a B cell-specific, matrix associating region-binding protein that transactivates gene expression from the IgH intronic enhancer (E mu). We show here that Bright has multiple contextual requirements to function as a transcriptional activator. Bright cannot transactivate via out of context, concatenated binding sites. Transactivation is maximal on integrated substrates. Two of the three previously identified binding sites in E mu are required for full Bright transactivation. The Bright DNA binding domain defined a new family, which includes SWI1, a component of the SWI.SNF complex shown to have high mobility group-like DNA binding characteristics. Similar to one group of high mobility group box proteins, Bright distorts E mu binding site-containing DNA on binding, supporting the concept that it mediates E mu remodeling. Transfection studies further implicate Bright in facilitating spatially separated promoter-enhancer interactions in both transient and stable assays. Finally, we show that overexpression of Bright leads to enhanced DNase I sensitivity of the endogenous E mu matrix associating regions. These data further suggest that Bright may contribute to increased gene expression by remodeling the immunoglobulin locus during B cell development.

PSF is a novel corepressor that mediates its effect through Sin3A and the DNA binding domain of nuclear hormone receptors.

Members of the type II nuclear hormone receptor subfamily (e.g., thyroid hormone receptors [TRs], retinoic acid receptors, retinoid X receptors [RXRs], vitamin D receptor, and the peroxisome proliferator-activated receptors) bind to their response sequences with or without ligand. In the absence of ligand, these DNA-bound receptors mediate different degrees of repression or silencing of gene expression which is thought to result from the association of their ligand binding domains (LBDs) with corepressors. Two related corepressors, N-CoR and SMRT, interact to various degrees with the LBDs of these type II receptors in the absence of their cognate ligands. N-CoR and SMRT have been proposed to act by recruiting class I histone deacetylases (HDAC I) through an association with Sin3, although they have also been shown to recruit class II HDACs through a Sin3-independent mechanism. In this study, we used a biochemical approach to identify novel nuclear factors that interact with unliganded full-length TR and RXR. We found that the DNA binding domains (DBDs) of TR and RXR associate with two proteins which we identified as PSF (polypyrimidine tract-binding protein-associated splicing factor) and NonO/p54(nrb). Our studies indicate that PSF is a novel repressor which interacts with Sin3A and mediates silencing through the recruitment of HDACs to the receptor DBD. In vivo studies with TR showed that although N-CoR fully dissociates in the presence of ligand, the levels of TR-bound PSF and Sin3A appear to remain unchanged, indicating that Sin3A can be recruited to the receptor independent of N-CoR or SMRT. RXR was not detected to bind N-CoR although it bound PSF and Sin3A as effectively as TR, and this association with RXR did not change with ligand. Our studies point to a novel PSF/Sin3-mediated pathway for nuclear hormone receptors, and possibly other transcription factors, which may fine-tune the transcriptional response as well as play an important role in mediating the repressive effects of those type II receptors which only weakly interact with N-CoR and SMRT.

Regulation of matrix attachment region-dependent, lymphocyte-restricted transcription through differential localization within promyelocytic leukemia nuclear bodies.

Bright (B cell regulator of IgH transcription) transactivates the immunoglobulin heavy chain (IgH) intronic enhancer, Emicro, by binding to matrix attachment regions (MARs), sites necessary for DNA attachment to the nuclear matrix. Here we report that Bright interacts with the ubiquitous autoantigen Sp100, a component of promyelocytic leukemia nuclear bodies (PML NBs), and with LYSp100B/Sp140, the lymphoid-restricted homolog of Sp100. Both in intact cells and in nuclear matrix preparations, the majority of Bright and Sp100 colocalize within PML NBs. In contrast, Bright colocalizes with only a small fraction of LYSp100B while inducing a redistribution of the majority of LYSp100B from its associated nuclear domains (LANDs) into nucleoplasm and cytoplasm. Sp100 represses the MAR-binding and transactivation activity of Bright. LYSp100B interacts more weakly with Bright but requires significantly higher levels than Sp100 to inhibit MAR binding. However, it strongly stimulates Bright transactivation through E mu. We suggest that Sp100 and LYSp100B interactions with Bright have different consequences for IgH transcription, potentially through differential association of E mu MARs with nuclear matrix- associated PML NBs and LANDs.

Positive and negative selection of antigen-specific B cells in transgenic mice expressing variant forms of the V(H)1 (T15) heavy chain.

Four variant forms of the V1 (T15-H chain) gene are synthesized in mice. Each V1 variant pairs with a distinct L chain to produce a binding site having specificity for phosphocholine (PC). Transgenic mice expressing variant forms of the V1 gene were analyzed to elucidate the factors driving B cell selection into the peripheral repertoire. In all four lines of H chain transgenic mice analyzed, transgene expression caused complete allelic exclusion of endogenous H chains in the bone marrow (BM), whereas most splenic B cells expressed endogenous H chains. The number of sIgM(+) BM B cells and their sIg receptor number was reduced compared to that of normal transgene-negative controls, suggesting that B cells expressing transgene-encoded H chains were being negatively selected in the BM. Mice expressing autoreactive forms of the V1 transgene with lower affinity for PC (M603H and M167H) exhibit positive selection of PC-specific B cells into the spleen, whereas mice expressing the higher affinity T15H variant exhibited elevated PC-specific B cells in the peritoneal cavity but few V(H)1(+) splenic B cells. These data suggest that the higher affinity T15-id(+) B cells preferentially survive in the peritoneal cavity. When these H chain transgenes were crossed into the mu MT knockout mouse in which surface expression of endogenous H chains is blocked, the percent of splenic V(H)1(+) PC-specific B cells increased up to 5-fold and T15-id(+) B cells were detectable in the spleen of T15H mice. This implies that T15-id(+) PC-specific B cells can be selected into the periphery, but they compete poorly with follicular B cells expressing endogenous Ig.

ARID proteins come in from the desert.

Members of the recently discovered ARID (AT-rich interaction domain) family of DNA-binding proteins are found in fungi and invertebrate and vertebrate metazoans. ARID-encoding genes are involved in a variety of biological processes including embryonic development, cell lineage gene regulation and cell cycle control. Although the specific roles of this domain and of ARID-containing proteins in transcriptional regulation are yet to be elucidated, they include both positive and negative transcriptional regulation and a likely involvement in the modification of chromatin structure.

Nuclear NonO/p54(nrb) protein is a nonclassical carbonic anhydrase.

The growing carbonic anhydrase (CA) gene family includes 11 enzymatically active isozymes in mammals. Each of them has a characteristic cellular and subcellular distribution pattern. In this report, we demonstrate for the first time a nuclear protein with CA activity. A polypeptide recognized by CA II antibodies was purified from several rat tissues using CA inhibitor affinity chromatography. This polypeptide of apparent 66 kDa mass was characterized using amino acid sequencing and CA activity measurements. It appeared to be identical to nonO/p54(nrb), a previously cloned and characterized RNA and DNA binding nuclear factor. Recombinant nonO generated in baculovirus bound to the CA inhibitor affinity chromatography matrix and revealed detectable CA activity (25 units/mg). Hansson's histochemical staining of rat lymph nodes followed by light and electron microscopy showed nuclear CA activity in lymphocytes, suggesting that the nuclear nonO protein is catalytically active in vivo. These results demonstrate that a previously known transcription factor is a novel, nonclassical CA. Through its CA activity, the nonO may function in the maintenance of pH homeostasis in the nucleus.

A plasma cell leukemia patient showing bialleic 14q translocations: t(2;14) and t(11;14).

We report here an IgG/lambda-type plasma cell leukemia patient showing bialleic 14q32 translocations. All immunoglobulins were suppressed in this patient, but a small amount of monoclonal IgG was detected by immunoelectrophoresis. Two cells of six peripheral blood mononuclear cells showed 46,XY,t(2;14)(q11;q32), i(8)(q10), t(11;14)(q13;q32), del(12)(q13.1) by karyotypic analysis. We confirmed the juxtaposition of IgH and PRAD1/Cyclin D1 genes by fluorescent in situ hybridization and overexpression of the PRAD1/Cyclin D1 gene, but Southern analysis showed no bcl-1 rearrangement. We analyzed the t(2;14)(q11;q32) using DNA fragments derived from childhood B-chronic lymphocytic leukemia cases bearing t(2;14)(p13;q32). Southern and Northern analyses demonstrated no alteration of these genes, indicating that this t(2;14) was different from that of childhood B-chronic lymphocytic leukemia. At the IgH loci, Southern analysis showed two rearranged bands and one germ-line band of JH. Cmicro was deleted on one rearranged allele but remained on the other, suggesting that the chromosome translocation occurred after productive class switch recombination on the Cmicro deleted allele.

Cux/CDP homeoprotein is a component of NF-muNR and represses the immunoglobulin heavy chain intronic enhancer by antagonizing the bright transcription activator.

Nuclear matrix attachment regions (MARs) flanking the immunoglobulin heavy chain intronic enhancer (Emu) are the targets of the negative regulator, NF-muNR, found in non-B and early pre-B cells. Expression library screening with NF-muNR binding sites yielded a cDNA clone encoding an alternatively spliced form of the Cux/CDP homeodomain protein. Cux/CDP fulfills criteria required for NF-muNR identity. It is expressed in non-B and early pre-B cells but not mature B cells. It binds to NF-muNR binding sites within Emu with appropriate differential affinities. Antiserum specific for Cux/CDP recognizes a polypeptide of the predicted size in affinity-purified NF-muNR preparations and binds NF-muNR complexed with DNA. Cotransfection with Cux/CDP represses the activity of Emu via the MAR sequences in both B and non-B cells. Cux/CDP antagonizes the effects of the Bright transcription activator at both the DNA binding and functional levels. We propose that Cux/CDP regulates cell-type-restricted, differentiation stage-specific Emu enhancer activity by interfering with the function of nuclear matrix-bound transcription activators.

Regulatory elements in the promoter of a murine TCRD V gene segment.

TCRD V segments rearrange in an ordered fashion during human and murine thymic development. Recombination requires the accessibility of substrate gene segments, and transcriptional enhancers and promoters have been shown to regulate the accessible chromatin configuration. We therefore investigated the regulation of TCRD V rearrangements by characterizing the promoter of the first TCRD V segment to be rearranged, DV101S1, under the influence of its own enhancer. Sequences required for full promoter activity were identified by transient transfections of normal and mutated promoters into a human gammadelta lymphoma, and necessary elements fall between -86 and +66 nt, relative to the major transcription start site. They include a cAMP responsive element (CRE) at -62, an Ets site at -39, a TATA box at -26, the major transcriptional start site sequence (-8 to -5 and -2 to +11), and a downstream sequence (+12 to +33). Gel shift analyses and in vitro DNase I footprinting showed that nuclear proteins bind to the functionally relevant CRE, Ets, +1 to +10 sequence, and the +17 to +21 sequence. Nuclear proteins also bind to an E box at -52, and GATA-3 binds to a GATA motif at -5, as shown by Ab ablation-supershift experiments, but mutations that abrogated protein binding to these sites failed to affect DV101S1 promoter activity. We conclude that not all protein-binding sites within the DV101S1 minimal promoter are important for enhancer driven TCRD gene transcription. Further, the possibility remains that the GATA and E box sites function in enhancer independent DV101S1 germline transcription.

Hybrid membrane IgM with the transmembrane region of I-A alpha facilitates enhanced presentation of distinct epitopes to T cells.

The role of B cell Ag receptors (membrane Ig or mIg) in the efficient Ag presentation to T cells, including the requirement of mIgM-associated Ig alpha/Igbeta, remains unclear. We report here that mIgM, substituted with greater than two-thirds of the NH2-terminal A alpha transmembrane (TM) regions of the MHC class II molecule, are capable of mediating the efficient presentation of specific Ag to some (Group 1) but not all (Group 2) T cell hybridomas. In contrast, the generation of epitopes recognized by the Group 2 hybridomas can be mediated only by the wild-type mIgM. Tyrosine phosphorylation appears to be necessary for the enhanced Ag presentation to Group 2 hybridomas, while it does not for Group 1 hybridomas. In addition, differential sensitivity of Ag processing to leupeptin, different duration required for epitope generation/presentation, as well as the involvement of distinct epitopes for stimulation of these groups of T cell hybridomas were observed. These results suggest that transport of the mIgM/Ag complexes to an endocytic compartment(s) for generation of certain T cell epitopes may be mediated by the N-terminal TM sequence of mIgM, independent of Ig alpha/Igbeta association. This function can be replaced by two-thirds of the NH2-terminal TM region of A alpha chain of class II molecules.

NonO enhances the association of many DNA-binding proteins to their targets.

NonO is an unusual nucleic acid binding protein not only in that it binds both DNA and RNA but that it does so via functionally separable domains. Here we document that NonO enhances the binding of some (E47, OTF-1 and OTF-2) but not all (PEA3) conventional sequence-specific transcription factors to their recognition sites in artificial substrates as well as in an immunoglobulin VHpromoter. We also show that NonO induces the binding of the Ku complex to DNA ends. Ku has no known DNA sequence specificity. These enhancement of binding effects are NonO concentration dependent. Using the E box activity of E47 as a model, kinetic studies demonstrate that the association rate of the protein-DNA complex increases in the presence of NonO while the dissociation rate remains the same, thereby increasing the sum total of the interaction. Oligo competition experiments indicate that NonO does not contact the target DNA in order to enhance the binding activity of DNA binding proteins. Rather, methylation interference analysis reveals that the induced E47 binding-activity has the same DNA-binding sequence specificity as the normal binding. This result suggests that one of the effects of NonO is to induce a true protein-DNA interaction. In this way, it might be possible for NonO to play a crucial role in gene regulation.

Ku is a general inhibitor of DNA-protein complex formation and transcription.

Ku is a ubiquitous and abundant DNA binding protein. Recently, it has been shown that Ku plays a crucial role in double stranded-DNA (dsDNA) break repair such as occurs during the V(D)J recombination of Ig genes. Ku has also been found to provide DNA binding activity to the catalytic domain of DNA-PK which is known to phosphorylate several transcription factors, suggesting that Ku is a multifunctional protein that participates as a component of several functional DNA-protein complexes. Here, we examined the interaction of Ku with several DNA binding proteins. Firstly, the DNA binding interaction between Ku and well-characterized transcription factors (OTF-1, Sp-1, AP-1) was analysed by EMSA. Although sequence non-specific, Ku was strongly competitive with these sequence specific transcription factors on compatible DNA elements, displacing them because of its high affinity association with DNA ends. Secondly, to determine whether this competitive effect was functionally relevant, we tested Ku in an in vitro transcription system with the adenovirus major late promoter. We found that Ku inhibited transcription from linear, but not from circular template DNA. These results suggest that Ku inhibits transcription when it is able to bind to template DNA and that the inhibition is the result of Ku displacing specific transcription factors from DNA.

A comparison of 2 analgesic regimens for the control of postoperative periodontal discomfort.

A single blind study of 24 patients compared the postoperative periodontal pain relief and adverse effects associated with a pretreatment regimen with etodolac, a nonsteroidal anti-inflammatory drug (NSAID), to a typical pro re nada (prn) regimen with a combination of acetaminophen with hydrocodone. Patients selected required one or more periodontal osseous surgeries that were judged to involve relatively similar degrees of surgical manipulation. Patients in the etodolac group received two 300 mg capsules 30 minutes prior to surgery and then redosed themselves prn. Patients who received the combination drug were not premedicated and followed a prn regimen. The subjects used a verbal analogue scale to report levels of pain hourly for the first 8 hours (starting 30 minutes prior to surgery) and also indicated any side effects experienced during the first week after surgery. Specific parameters monitored were the mean sum of hourly pain scores, mean hourly pain scores, time to first medication, number of postoperative doses, and adverse effects. Of the parameters studied, the only one that showed a statistically significant difference was the time to first medication. The time span from 30 minutes prior to the beginning of surgery to the first postsurgical dose was greater for etodolac than for the combination drug. However, the total number of medications taken under both regimens was similar. The side effects were minimal for both of the drugs studied. It was concluded that the analgesic regimens tested under clinical practice conditions were comparable in providing analgesia with minimum side effects in uncomplicated periodontal osseous surgery. Studies with larger numbers of patients are needed to definitively address whether these regimens are truly equivalent.

Analysis and comparison of the mouse and human immunoglobulin heavy chain JH-Cmu-Cdelta locus.

We report here 23,686 bases of contiguous DNA sequences from the mouse germline immunoglobulin heavy chain (H) constant (C) mu delta region. The sequence spans the joining (JH) regions, the mu constant region (C mu), the delta constant region (C delta) coding regions, a domain relic, the mu switch region (S mu), seven blocks of simple sequence repeats, a large unique sequence inverted repeat, a large unique sequence forward repeat, and all of the intervening material. A comparison of this 23.7-kb region with the corresponding human C mu/C delta region reveals clear homology in the coding and introns of C mu but not in the 5' flanking J gene segments nor in the intergenic and C delta regions. This mixed pattern of similarity between the human and the mouse sequences contrasts with high levels of similarity found in the T-cell receptor C alpha/C delta region and alpha and beta myosin genes and the very low levels found in the gamma-crystallin, XRCC1, and beta-globin gene clusters. The human and mouse comparison further suggests the incorporation of novel sequences into expressed genes of IgD.

The immunoglobulin heavy-chain matrix-associating regions are bound by Bright: a B cell-specific trans-activator that describes a new DNA-binding protein family.

B lymphocyte-restricted transcription of immunoglobulin heavy-chain (IgH) genes is specified by elements within the variable region (VH) promoter and the intronic enhancer (E mu). The gene encoding a protein that binds a VH promoter proximal site necessary for induced mu-heavy-chain transcription has been cloned. This B-cell specific protein, termed Bright (B cell regulator of IgH transcription), is found in both soluble and matrix insoluble nuclear fractions. Bright binds the minor groove of a restricted ATC sequence that is sufficient for nuclear matrix association. This sequence motif is present in previously described matrix-associating regions (MARs) proximal to the promoter and flanking E mu. Bright can activate E mu-driven transcription by binding these sites, but only when they occur in their natural context and in cell lines permissive for E mu activity. To bind DNA, Bright requires a novel tetramerization domain and a previously undescribed domain that shares identity with several proteins, including SWI1, a component of the SWI/SNF complex.