A single 3' alpha hs1,2 enhancer in the rabbit IgH locus

Multiple cis-acting elements including the intronic enhancer and the 3'alpha enhancer (3'alphaE) regulate expression of the Ig heavy chain genes during B cell development. A 3'alphaE is composed of DNase I-hypersensitive sites, hs1,2, hs3a,b, and hs4, found 3' of the murine Calpha gene as well as 3' of both human Calpha genes, Calpha1 and Calpha2. Rabbits have 13 Calpha genes, and we tested whether a 3'alphaE is associated with each of these genes. To identify 3'alphaE regions we developed a rabbit hs1,2 probe and used this to search for enhancer homologues of human hs1,2 in a genomic fosmid library. We identified a single hs1,2 fragment 8-kb downstream of Calpha13, the presumed 3'-most Calpha gene. We also identified and partially sequenced a new Calpha gene, Calpha14, located 6 kb upstream of Calpha13. Genomic Southern blot analysis confirmed that the rabbit genome contains only one hs1,2 enhancer region. We tested the enhancer activity of the hs1,2 with the SV40, V(H), and Ialpha promoters using the luciferase reporter gene in transient transfection assays and found that it significantly enhanced the activity of SV40 and V(H) promoters and slightly enhanced an Ialpha promoter. We conclude that the rabbit has a single hs1,2 enhancer that resides at the 3' end of the IgH gene cluster and may constitute one of the cis-elements regulating the expression of IgH genes.     

Hepatitis B virus (HBV) promoters are regulated by the HBV enhancer in a tissue-specific manner.

The activities of the individual hepatitis B virus (HBV) promoters and the effects of the HBV enhancer on these promoters in several human cell types have been compared by measuring the activity and RNA levels of the linked reporter function chloramphenicol acetyltransferase. The relative promoter activities in the human HepG2 (liver), HeLa, and HS27 (fibroblast) cell lines are in the order precore greater than X greater than preS2 greater than preS1; thus, the promoters of the gene producing the largest quantity of viral proteins have relatively low activity. The juxtaposition of the HBV enhancer in either orientation increased the promoter activities only modestly (2- to 5-fold) in the nonliver cell lines, whereas it dramatically increased (20- to 100-fold) the promoter activities in the liver cell line. Thus, the HBV enhancer is especially active in liver cells. This may be one of the causes of hepatotrophicity of the virus.     

Evaluation of novel control elements by construction of eukaryotic expression vectors

A novel mammalian eukaryotic expression vector for the production of immunoglobulin heavy chain (IgH) genes has been designed. This expression vector contains the variable heavy chain (VH) promoter, the IgH intron enhancer (μE) and the IgH 3′ enhancer (3′E). This construct, designated pTIF-1, was stably transfected into the myeloma cell line J558L. A fivefold increase in the expression level of a rearranged IgH gene was observed when using the pTIF-1 vector containing the 3′E compared to an expression vector lacking this enhancer. Interestingly, this positive effect on the expression level of the 3′ enhancer appears to be position independent. The introduction of two recently identified Ig control elements, HS3 and HS4, to the vector cassette did not further elevate the expression level in the cell line tested. The pTIF-1 vector can be used for expression of any antibody specificity, using PCR amplification of the VDJ region of interest. Furthermore, the constant region can easily be exchanged, which further facilitates studies to dissect different effector functions of IgH constant genes.     

Nuclear factors binding to the human immunoglobulin heavy-chain gene enhancer.

The human immunoglobulin heavy chain (IgH) gene contains at least two tissue-specific regulatory regions, which are similar to the mouse IgH gene. One is the J-C enhancer and another is located in the 5' promoter region. Using an electrophoretic mobility shift assay and DNase I footprint, we have examined the interaction of factors in B cell nuclear extracts with the two regulatory regions of the human IgH gene. We have identified a nuclear factor in mouse B cell nuclear extracts which bound to specific sequence in the human IgH enhancer. This factor is apparently not present in mouse fibroblast nuclear extracts. We also found factor(s) which bound to the highly conserved octanucleotide sequence within the human IgH enhancer and 5' promoter regions.     

IgM receptor-mediated transactivation of the IgH 3' enhancer couples a novel Elf-1-AP-1 protein complex to the developmental control of enhancer function.

The function of the temporally regulated B lymphocyte-specific immunoglobulin heavy chain (IgH) 3' enhancer has been linked to the IgH class switch machinery, but the physiological mechanism(s) of activation has not been discerned. Following crosslinking of the IgM receptor, we demonstrate that the enhancer is transactivated in the B lymphoma cell line BAL-17. In both induced primary B lymphocytes and BAL-17 cells, the enhancer activation is concomitant with the recruitment of a novel DNA binding complex, nuclear factor of activated B cells (NFAB). NFAB contains the tissue-restricted Ets protein Elf-1 and the AP-1 factors Jun-B and c-Fos, which bind to a novel 3' enhancer ETS-AP-1 motif. IgM receptor-mediated activation or stimulation by phorbol-ester in BAL-17 cells demonstrates that the ETS-AP-1 motif, when linked to a heterologous gene, can confer a ligand/receptor-dependent response. In NIH 3T3 cells, Elf-1 expression is required for efficient ETS-AP-1 promoter activity in response to stimulation by 12-O-tetradecanylphorbol 13-acetate. Our results suggest a biological role for Elf-1 in the regulation of IgH gene expression, attribute a functional role for receptor-induced AP-1 proteins in B lymphocytes and provide evidence for a direct link between IgM receptor-mediated signalling and 3' enhancer activation.     

DNase I-defined chromatin configuration of the human CD3 gene cluster.

The three CD3 genes on human chromosome 11q23 encode proteins (gamma, delta and epsilon) which form part of the antigen receptor on T lymphocytes. All three genes are clustered within 50 kb and are activated approximately contemporaneously during the early stages of T cell ontogeny. In order to pinpoint potential regulatory sequences important for locus activation and tissue-specific gene expression, the chromatin structure of almost 90 kb of this region has been probed in five cell lines using the endonuclease pancreatic DNase I. A set of DNase I hypersensitive (HS) sites has been defined in T cell chromatin, five of which were strong and not found in non-T cells, with the exception of the erythroleukaemia cell line K562, in which three sites were weakly expressed, correlating with a low level of delta mRNA. The subset of five HS sites map close to the CD3 genes and lie in regions which contain elements of defined function: the gamma promoter; the delta promoter and its 3' enhancer; and the epsilon promoter and its 3' enhancer. Since no further major T cell-restricted HS sites lie within the 90kb of the CD3 locus analysed, these five regions may contain all the sequences important for CD3 gene expression.