In vivo and in vitro analysis of the human tissue-type plasminogen activator gene promoter in neuroblastomal cell lines: evidence for a functional upstream kappaB element.

Besides its well-established role in wound healing and fibrinolysis, tissue-type plasminogen activator (t-PA) has been shown to contribute to cognitive processes and memory formation within the central nervous system, and to promote glutamate receptor-mediated excitotoxicity. The t-PA gene is expressed and regulated in neuronal cells but the regulatory transcriptional processes directing this expression are still poorly characterized. We have used DNase I-hypersensitivity mapping and in vivo foot printing to identify putative regulatory elements and transcription factor binding sites in two human neuroblastomal (KELLY and SK-N-SH) and one human glioblastomal (SNB-19) cell lines. Hypersensitive sites were found in the proximal promoter region of all cell lines, and within the first exon for KELLY and SNB-19 cells. Mapping of methylation-protected residues in vivo detected a cluster of protected residues corresponding to a cAMP response element (CRE) and Sp1 sites in the proximal promoter previously shown to be essential for basal expression in other cell types. Protected residues were also found at other sites, notably a kappaB element at position bp -3081 to -3072 that was partly protected in KELLY and SNB-19 cells. Analysis of transfected reporter constructs in KELLY and SNB-19 cells confirmed that this particular element is functionally significant in the transactivation of the t-PA promoter in both cell types. This study defines, by in vivo and in vitro methods, a previously undescribed kappaB site in the t-PA gene promoter that influences t-PA expression in neuronal cells.

Evolutionary dynamics of the human immunoglobulin kappa locus and the germline repertoire of the Vkappa genes.

We have determined the entire nucleotide sequence of the human immunoglobulin kappa locus, comprising a total of 1,010,706 nucleotides. The 76 Vkappa genes found by a hybridization-based approach and their classification in 7 families were confirmed. A Vkappa orphon located near the locus was also sequenced. In addition, we identified 55 novel Vkappa relics and truncated pseudogenes, which establish 5 new families. Among these 132 Vkappa genes, 46 have open reading frames. According to the databases and the literature, 32 unique Vkappa genes and 5 identical gene pairs form VJ-joints, 27 unique genes and 4 gene pairs are transcribed, and 25 unique genes and 4 gene pairs produce functional proteins. The Vkappa gene locus contains a 360-kb inverted duplication, which harbors 118 Vkappa genes. A comparison of the duplicated Vkappa genes suggests positive selection on the complementarity-determining regions of the duplicated genes by point mutations. The entire duplication unit was divided into 13 blocks, each of which has its distinct nucleotide sequence identity to its duplication counterpart (98.1 - 99.9 %). An inversion-mediated mechanism is suggested to generate the high-homology blocks. Based on the homology blocks and the mutation rates, the inverted duplication is assumed to have taken place approximately 5 million years ago. An orphon Vkappa gene near the kappa locus and a cluster of five Vkappa orphons on chromosome 22 have no counterparts within the kappa locus. This suggests possible mechanisms of the transposition of orphon Vkappa genes.

The immunoglobulin lambda light chain enhancer consists of three modules which synergize in activation of transcription.

V(D)J rearrangement, high level expression and somatic hypermutation of assembled Ig genes is tightly controlled by a number of regulatory sequence elements located in the vicinity of the J-, (D)-, and C-gene segments. During B cell maturation these elements become accessible to binding of trans-acting factors, reflecting the opening of the chromatin structure through an as yet unidentified mechanism. The mapping of regions of an altered chromatin structure (DNase I hypersensitivity) therefore is a powerful approach in identifying regulatory sequence elements. We here show that the human Ig lambda enhancer consists of three modules previously identified by us as DNase I-hypersensitive sites HSS-1, -2, and -3. The three sequence elements synergize in transcriptional activation of a reporter gene and together constitute a powerful tissue-specific enhancer which is a much stronger transcriptional activator than the kappa enhancers alone or in combination. We further show that the accessibility of the kappa and lambda enhancer elements for DNase I in the chromatin of a pre-B cell line (207) correlates with the transcriptional enhancer activities of kappa and lambda enhancer constructs. This finding is in support of an ordered model for Ig light chain activation (kappa before lambda).

Immunophenotypic features and configuration of immunoglobulin genes in hairy cell leukemia-Japanese variant.

Immunophenotypes and Ig gene rearrangements were investigated in 12 patients with a variant form of hairy cell leukemia (HCL) termed HCL-Japanese variant (HCL-J), and in an HCL-J-derived cell line. The leukemic cells of HCL-J characteristically showed the phenotype of CD20+, CD5-, CD10-, CD11c+, CD22+, CD24- and CD25-. Ig light (L) chain was undetected in nine cases, and the remaining four cases expressed kappa chain. Expression of Ig heavy (H) chain was studied in nine cases. In addition to Igkappa+ cases showing expression of predominantly gamma H chain isotype, alpha chain was detected in one case without expression of L chain. Rearranged bands in Ig heavy chain (JH) genes were recognized in all 12 cases tested. Rearranged bands in kappa chain genes and germline configuration in chi chain genes were seen in all three Igkappa+ cases tested. Four of nine cases without expression of L chain had a rearranged chi chain gene. The other three cases had chi chain genes in the germline configuration and rearranged and/or deleted kappa chain genes. In the remaining two cases, no rearrangement in either kappa or chi chain genes was detected. The Ig gene configuration and expression in HCL-J, partially overlapping with those described for immature B cell leukemia, were dissociated from the cytological features and CD20+, membrane CD22+ phenotype characteristic of mature B cells.

Tissue-specific deoxyribonuclease I-hypersensitive sites in the vicinity of the immunoglobulin C lambda cluster of man.

During B cell development, the onset of DNA rearrangements, expression, and somatic hypermutation of Ig genes are regulated through the complex interaction of cis-acting elements with trans-acting factors. Our aim is to identify DNA elements required during activation of the human Ig lambda light chain genes. Determination of deoxyribonuclease (DNase) I-hypersensitive sites in complex regulated genes can lead to the identification of sequence elements which would have been overlooked by employing transient transfection protocols. We have therefore investigated the chromatin structure of human J-C lambda genes and identified three DNase I-hypersensitive sites (HSS-1, -2, and -3) within an 8-kb region downstream of the J-C lambda 7 gene. HSS-2 and HSS-3 are B cell specific. The DNase I-hypersensitive sites are also present in kappa-producing cell lines which have not rearranged the Ig lambda locus and produce germ-line J-C lambda transcripts. We conclude that in mature B cells, both kappa and lambda loci are in an active structure regardless of the type of light chain they produce. This suggests that the chromatin structure of both loci is opened early in B cell development and that the active structure persists in mature B cells. The observed temporal order (first kappa, then lambda) of activation can be explained by consecutive synthesis of the appropriate regulating factors and the tight regulation of the recombination machinery through the products of L chain gene rearrangements.

The human replacement histone H3.3B gene (H3F3B).

H3.3 is a replacement histone subtype that is encoded by two replication-independent genes termed H3.3A and H3.3B, respectively. We have isolated a fullsize H3.3 cDNA clone from an oligo(dT)-primed human testicular cDNA library. Subsequently, the corresponding gene was isolated from a human cosmid library and was identified as the H3.3B gene. It was the only histone gene on this 42-kb cosmid clone. The gene structure shows characteristic features of an H3.3 gene. First, it contains an intron of about 0.5 kb in the 5' untranslated region and two smaller introns within the coding gene portion. Second, no histone gene-specific dyad symmetry element was found in the 3' untranslated region, but three putative polyadenylation signals were detected downstream of the gene. The corresponding transcripts were detected by Northern blot analysis using poly(A)+ RNA from testis and from the HEK293 tumor cell line. The newly discovered human H3.3B gene (HGMW-approved symbol H3F3B) was mapped by fluorescence in situ hybridization to the telomeric region of chromosome 17 (17q25). This localization of the H3.3B gene and its solitary arrangement contrast with the majority of the replication-dependent histone genes, which form a large cluster on chromosome 6 and a second cluster on chromosome 1.

Differential interactions between the immunoglobulin heavy chain mu intron and 3' enhancer.

The immunoglobulin heavy chain 3' enhancer may be a novel type of a transcriptional regulation element in as much as its function is position dependent. We show that there are interactions between the mu intron and 3' enhancer which are differentially regulated depending on the distance between the two elements. Thus, a transcriptional repression is exerted by the 3' enhancer when juxtaposed to the intron enhancer. Whereas no or only modest synergism between the immunoglobulin mu intron and 3' enhancer has been reported to date, we show here that the stimulatory effect is substantially increased by extending the distance between the two enhancers. In our stable expression system, the mu intron enhancer insulated the test gene from neighboring chromatin.

Ongoing V kappa-J kappa recombination after formation of a productive V kappa-J kappa coding joint.

V kappa genes of man can recombine with the J kappa gene segments either by an inversion or by a deletion mechanism. Back-to-back fusion products of the respective recombination signal sequences (signal joints) are retained on the chromosome after the formation of a V kappa-J kappa coding joint by an inversion. Our knowledge of the structure of the human kappa locus and the application of the polymerase chain reaction allowed us now to establish a direct relationship between different kappa recombination products in the lymphoid cell line JI. Two consecutive inversions fully explain the existence of two coding joints and two signal joints on the same chromosome of this cell line. Although the initially formed coding joint is productively rearranged and expressed, a second V kappa-J kappa rearrangement took place which leads to an aberrant joint. In this process a J kappa gene segment of the signal joint that had been created in the first V kappa-J kappa joining was used as the recombination target. The sequence of the two rearrangements is unequivocal since a product of the first (productive) reaction is a partner in the second (aberrant) one.

V lambda and J lambda-C lambda gene segments of the human immunoglobulin lambda light chain locus are separated by 14 kb and rearrange by a deletion mechanism.

We have cloned a region of 124 kb of the human immunoglobulin lambda light chain locus on chromosome 22 encompassing seven V lambda and seven J-C lambda gene segments. No further C lambda gene segment was found in a region of 35 kb downstream of C lambda 7, which encodes the Ke+Oz- isotype. The C lambda proximal V lambda gene segment V lambda III. 1 is located 14.5 kb upstream of C lambda 1. The five sequenced V lambda genes have the same transcriptional orientation as the J-C lambda gene segments which is likely to be true for the majority of the V lambda gene segments in the human lambda locus and which suggests a deletion mechanism for DNA rearrangement. This is supported by hybridization of V lambda gene probes to germ-line and rearranged DNA from lambda light chain-producing cell lines. Sequences of 23 cDNA clones allow to establish a V lambda subgroup classification based on nucleic acid sequence data and an estimate of the J-C lambda usage.

Megabase inversions in the human genome as physiological events.

The genes of the immunoglobulin kappa light chains are assembled during B-cell differentiation by somatic recombination of one of the V kappa (variable) gene segments and the J kappa-C kappa (joining-constant) gene region. This seems to occur by deletion of the DNa between V kappa and J kappa-C kappa if they are arranged in germ-line DNA in the same transcriptional polarity or by inversion of a fragment containing the V kappa gene if the polarities are opposite. We have cloned 75 V kappa genes and pseudogenes of the human kappa locus and linked them in large contigs. There seem to be no more than 85 such genes, less than 50 of these being potentially functional. Thirty-eight of the cloned genes have the same transcriptional polarity as J kappa-C kappa and are part of the so-called J kappa proximal cluster; 35 genes in a distal cluster (the result of a duplication event in evolution) have a polarity that was suggested to be opposite to the one of J kappa-C kappa. We now show that the V kappa genes of the proximal cluster rearrange by a deletion mechanism whereas the others join J kappa-C kappa by inversion of megabase-sized DNA fragments.

The V kappa gene repertoire in the human germ line.

The question of how many V kappa gene segments exist in the human germ line was addressed. Seventy-five V kappa genes of the kappa locus and twenty-five V kappa genes localized outside of the locus ("orphons") had been cloned previously; 67 of the genes and 19 of the orphons had already been sequenced yielding 36 and 1 potentially functional V kappa genes, respectively, the remaining ones being pseudogenes. We now (a) determined the relative hybridization intensities of the cloned V kappa genes and orphons, (b) identified the bands in blot hybridizations of genomic DNA digests with the cloned genes and orphons, (c) determined the band intensities in the genomic DNA digests from two individuals and one cell line, (d) normalized the results with the help of the C kappa gene segment which is present in the haploid genome in one copy, (e) compared the genomic blot hybridization patterns with patterns of equimolar mixtures of the cloned V kappa genes and orphons, and (f) defined the bands and fractional intensities in bands that could not be assigned to cloned genes or orphons. From the resulting data we conclude that there are 5-7 still uncloned V kappa genes in germ-line DNA in addition to the 75 known V kappa genes and in addition to the 25 orphons 12-15 orphon candidates. It appears that the rheumatoid factor light chains of the Wa and 6B6.6 idiotypes are coded for by one V kappa III gene each. It is concluded that the kappa locus comprises no more than 50 potentially functional genes and no more than 85 V kappa genes altogether.

The synthetic androgen mibolerone induces transient suppression of the transformed phenotype in an androgen responsive human prostatic carcinoma cell line.

The synthetic androgen mibolerone elicits a set of distinct changes in the behaviour of an androgen responsive human prostatic carcinoma cell line (LNCaP). Inhibition of cell proliferation, induction of morphological change and of a prostate specific mRNA, and inhibition of colony formation in soft agar are induced by very low concentrations of mibolerone. The natural androgen dihydrotestosterone is much less effective. The changes in growth characteristics and morphology are reverted by excess antiandrogen, i.e. cyproterone acetate or hydroxyflutamide. Cell lines lacking androgen receptors (PC-3, DU 145 and MRC-5) are completely unresponsive to mibolerone. Taken together, our results indicate androgen receptor mediated suppression of the transformed phenotype in LNCaP cells.

The V-J-intergenic region of the human kappa locus.

The 23-kb region between the V kappa and J kappa gene clusters was investigated in some detail. The region was found to be free of V kappa genes or V kappa gene-like structures, confirming the previous supposition that the V kappa gene B3 is the J kappa proximal V kappa gene. The B3-J kappa distance of 23 kb was found to be the same in the DNAs of several individuals. A HindIII restriction fragment length polymorphism was detected within this region. A sequence of 533 bp located approximately in the middle of the region has a highly homologous counterpart (called homox) on another chromosome. The two sequences are 96% identical. Possible mechanisms for the generation of such a duplicate are discussed.

Linking of the human immunoglobulin VK and JKCK regions by chromosomal walking.

The linking of the human VK and JKCK gene regions (abbreviations in ref. 1) by chromosomal walking is reported. Hybridization experiments with the DNA of a somatic cell hybrid containing the region between JKCK and the telomer show that none of the major VK gene clusters is located downstream of CK. The distance between the VK and JK genes was found to be 23 kb. The JK proximal VK gene is the B3 gene which is the only representative of subgroup IV in the genome. This gene and the neighbouring B2 gene (accompanying paper) are arranged in opposite orientation to JKCK and can therefore rearrange only by an inversion mechanism. This finding is used, together with previous data, to delineate the rearrangement processes in the Burkitt lymphoma derived cell line BL21 as comprising an inversion in the first and a deletion in the second step.

Studies on the immunoglobulin V kappa locus in human lymphoid cell lines.

DNA digests of 16 human lymphoid cell lines were studied in blot hybridization experiments with probes from V kappa genes and their immediate neighborhood as well as with single or low-copy probes from intergenic regions. The patterns were compared with those of placenta DNA digests in which the kappa genes are in the germline configuration. The differences of patterns which were detected with the first type of hybridization probes can be attributed to V kappa--J kappa rearrangements or to restriction site polymorphisms between individuals. Some of the pattern differences observed with the second type of probes can be interpreted best as arising from deletions of parts of the kappa locus. Such deletions may be individual variations but they may also be caused by the V kappa-J kappa rearrangement process. The results obtained with one particular probe which was derived from a nonduplicated part of the kappa locus allow some conclusions as to the mechanism of the V kappa--J kappa rearrangement: the genomic situation in some lymphoid cell lines can be explained by an inversion while in other cell lines clearly deletions have occurred. The observations are in agreement with the inversion-deletion mechanism of V kappa--J kappa rearrangement as proposed by Lewis et al. (1982, 1984).