Nuclear transcripts of mouse heavy chain immunoglobulin genes contain only the expressed class of C-region sequences.

In plasmacytoma cells producing IgG, IgA, or IgM immunoglobulin heavy chains, the large precursors of the heavy chain messenger RNA's contain nucleotide sequences that specify only the expressed class of constant region. This indicates that the switch from one class of heavy chain to another during B cell ontogeny does not occur by altered processing of a complex gene transcript.

Structure of the constant and 3' untranslated regions of the murine gamma 2b heavy chain messenger RNA.

The complete coding sequence for the constant region of the mouse gamma 2b immunoglobulin heavy chain and the 3' untranslated region has been determined. The coding portion of the sequence is 1008 nucleotides long (amino acid residues 114 to 449), and the 3' noncoding region contains 102 nucleotides preceeding the polyadenylate. An extra carboxyl-terminal lysine residue which had not been observed in the gamma 2b or other gamma subclass protein sequences occurs in the nucleotide sequence and is probably processed posttranslationally. A 17-nucleotide sequence occurs with slight variation twice in CH1 and once in CH2 domains in the same relative location but with different translational phase. This sequence may be the site of crossover in a gamma 2b . gamma 2a heavy chain variant, an indication of possible recombinational activity of some kind.

Sequence of the cloned gene for the constant region of murine gamma 2b immunoglobulin heavy chain.

The complete nucleotide sequence of the gamma 2b constant region gene cloned from BALB/c liver DNA is reported. The sequence of approximately 1870 base pairs includes the 5' flanking, 3' untranslated, and 3' flanking regions and three introns. The C gamma 2b coding region is divided by these introns into four segments corresponding to the homology domains and hinge region of the protein. The introns separating the hinge from the CH2 domain and the CH2 from the CH3 domain are small (106 and 119 base pairs). A larger intervening sequence of 314 base pairs separates the CH1 and hinge regions. The stretch of DNA comprising this large intron plus the hinge shows a strong homology with the other CH domains.

Transcription of germ line V alpha segments correlates with ongoing T-cell receptor alpha-chain rearrangement.

M14T is a virally transformed immature T-cell line which continues to rearrange its T-cell antigen receptor (TCR) alpha-chain genes in vitro and thus represents a dynamic system for studying TCR assembly. In an effort to investigate whether the TCR alpha locus is accessible for V(D)J rearrangement events, we examined M14T cells for the presence of germ line TCR alpha transcripts. Several unrearranged V alpha segments were found to be transcriptionally active in M14T cells. By comparison, germ line V alpha transcripts are absent in nonlymphoid and pro-T-cell lines and barely detectable in mature T-cell lines, suggesting that this phenomenon is likely stage and tissue specific. We demonstrate a perfect correlation between transcriptionally active V alpha segments and their involvement in ongoing V alpha-to-J alpha rearrangements. In addition, data suggesting that the unrearranged J alpha locus is also transcriptionally active in the M14T line are presented. Furthermore, the recombination-activating genes RAG-1 and RAG-2 are differentially expressed, with RAG-2 detectable only by polymerase chain reaction, implying that very low levels of one of these gene products are sufficient to complement the other to facilitate VJ rearrangements. These findings provide the first direct evidence for an accessibility model of antigen receptor rearrangement in T cells.

Structure of germ line immunoglobulin alpha heavy-chain RNA and its location on polysomes.

We describe the structure of the major germ line RNA transcribed from unrearranged immunoglobulin alpha heavy-chain genes in immunoglobulin M-expressing cells of the I.29 mu B-cell lymphoma, a cell line capable of switching to immunoglobulin A expression upon lipopolysaccharide treatment. This germ line alpha RNA has a small open reading frame that does not include the C alpha domain, and this RNA appears to be present on polysomes in I.29 mu cells.

Rearrangements and deletions of immunoglobulin heavy chain genes in the double-producing B cell lymphoma I.29.

The B cell lymphoma I.29 consists of a mixture of cells expressing membrane-bound immunoglobulin M (IgM) (lambda) and IgA (lambda) of identical idiotypes. Whereas most of the cells express either IgM or IgA alone, 1 to 5% of the cells in this tumor express IgM and IgA simultaneously within the cytoplasm and on the cell membrane (R. Sitia et al., J. Immunol. 127:1388-1394, 1981; R. Sitia, unpublished data). When IgM+ cells are purified from the lymphoma and passaged in mice or cultured, a portion of the cells convert to IgA+. These properties suggest that some cells of the I.29 lymphoma may undergo immunoglobulin heavy chain switching, although it is also possible that the mixed population was derived by a prior switching event in a clone of cells. We performed Southern blotting experiments on genomic DNAs isolated from populations of I.29 cells containing variable proportions of IgM+ and IgA+ cells and on a number of cell lines derived from the lymphoma. The results were consistent with the deletion model for heavy chain switching, as the IgM+ cells contained rearranged mu genes and alpha genes in the germ line configuration on both the expressed and nonexpressed heavy chain chromosomes, whereas the IgA+ cells had deleted both mu genes and contained one rearranged and one germ line alpha gene. In addition, segments of DNA located within the intervening sequence 5' to the mu gene, near the site of switch recombination, were deleted from both the expressed and the nonexpressed chromosomes. Although mu genes were deleted from both chromosomes in the IgA+ cells, the sites of DNA recombination differed on the two chromosomes. On the expressed chromosome, Smu sequences were recombined with S alpha sequences, whereas on the nonexpressed chromosome, Smu sequences were recombined with S gamma 3 sequences.

A Myc-associated zinc finger protein binding site is one of four important functional regions in the CD4 promoter.

The CD4 promoter plays an important role in the developmental control of CD4 transcription. In this report, we show that the minimal CD4 promoter has four factor binding sites, each of which is required for full function. Using biochemical and mutagenesis analyses, we determined that multiple nuclear factors bind to these independent sites. We determined that an initiator-like sequence present at the cap site and an Ets consensus sequence are required for full promoter function. We also demonstrate that the Myc-associated zinc finger protein (MAZ) appears to be the predominant factor binding to one of these sites. This last site closely resembles the ME1a1 G3AG4AG3 motif previously shown to be a critical element in the P2 promoter of the c-myc gene. We therefore believe that the MAZ transcription factor is also likely to play an important role in the control of developmental expression of the CD4 gene.

Functional isoforms of IkappaB kinase alpha (IKKalpha) lacking leucine zipper and helix-loop-helix domains reveal that IKKalpha and IKKbeta have different activation requirements.

The activity of the NF-kappaB family of transcription factors is regulated principally by phosphorylation and subsequent degradation of their inhibitory IkappaB subunits. Site-specific serine phosphorylation of IkappaBs by two IkappaB kinases (IKKalpha [also known as CHUK] and IKKbeta) targets them for proteolysis. IKKalpha and -beta have a unique structure, with an amino-terminal serine-threonine kinase catalytic domain and carboxy-proximal helix-loop-helix (HLH) and leucine zipper-like (LZip) amphipathic alpha-helical domains. Here, we describe the properties of two novel cellular isoforms of IKKalpha: IKKalpha-DeltaH and IKKalpha-DeltaLH. IKKalpha-DeltaH and IKKalpha-DeltaLH are differentially spliced isoforms of the IKKalpha mRNA lacking its HLH domain and both its LZip and HLH domains, respectively. IKKalpha is the major RNA species in most murine cells and tissues, except for activated T lymphocytes and the brain, where the alternatively spliced isoforms predominate. Remarkably, IKKalpha-DeltaH and IKKalpha-DeltaLH, like IKKalpha, respond to tumor necrosis factor alpha stimulation to potentiate NF-kappaB activation in HEK293 cells. A mutant, catalytically inactive form of IKKalpha blocked IKKalpha-, IKKalpha-DeltaH-, and IKKalpha-DeltaLH-mediated NF-kappaB activation. Akin to IKKalpha, its carboxy-terminally truncated isoforms associated with the upstream activator NIK (NF-kappaB-inducing kinase). In contrast to IKKalpha, IKKalpha-DeltaLH failed to associate with either itself, IKKalpha, IKKbeta, or NEMO-IKKgamma-IKKAP1, while IKKalpha-DeltaH complexed with IKKbeta and IKKalpha but not with NEMO. Interestingly, each IKKalpha isoform rescued HEK293 cells from the inhibitory effects of a dominant-negative NEMO mutant, while IKKalpha could not. IKKalpha-DeltaCm, a recombinant mutant of IKKalpha structurally akin to IKKalpha-DeltaLH, was equally functional in these assays, but in sharp contrast, IKKbeta-DeltaCm, a structurally analogous mutant of IKKbeta, was inactive. Our results demonstrate that the functional roles of seemingly analogous domains in IKKalpha and IKKbeta need not be equivalent and can also exhibit different contextual dependencies. The existence of cytokine-inducible IKKalpha-DeltaH and IKKalpha-DeltaLH isoforms illustrates potential modes of NF-kappaB activation, which are not subject to the same in vivo regulatory constraints as either IKKalpha or IKKbeta.

Synergism of v-myc and v-Ha-ras in the in vitro neoplastic progression of murine lymphoid cells.

Murine bone marrow was either singly or doubly infected with retroviral vectors expressing v-myc (OK10) or v-Ha-ras. The infected bone marrow was cultured in a system that supports the long-term growth of B-lineage lymphoid cells. While the v-myc vector by itself had no apparent effect on lymphoid culture establishment and growth, infection with the v-Ha-ras vector or coinfection with both v-myc and v-Ha-ras vectors led to the appearance of growth-stimulated cell populations. Clonal pre-B-cell lines stably expressing v-Ha-ras alone or both v-myc and v-Ha-ras grew out of these cultures. In comparison with cell lines expressing v-Ha-ras alone, cell lines expressing both v-myc and v-Ha-ras grew to higher densities, had reduced dependence on a feeder layer for growth, and had a marked increase in ability to grow in soft-agar medium. The cell lines expressing both oncogenes were highly tumorigenic in syngeneic animals. These experiments show that the v-myc oncogene in synergy with v-Ha-ras can play a direct role in the in vitro transformation of murine B lymphoid cells.

A cis-acting element in the promoter region of the murine c-myc gene is necessary for transcriptional block.

A block to elongation of transcription has been shown to occur within the first exon of the human and murine c-myc genes. The extent of this block was found to vary with the physiological state of cells, indicating that modulation of the transcriptional block can serve to control the expression of this gene. To determine which sequences are required in cis for the transcriptional block, we generated a series of constructs containing various portions of murine c-myc 5'-flanking and exon 1 sequences. We established populations of HeLa and CV-1 cells stably transfected with these constructs. The transcription start sites were determined by S1 nuclease mapping analysis, and the extent of transcriptional block was measured by nuclear run-on transcription assays. Our results demonstrate that at least two cis-acting elements are necessary for the transcriptional block. A 3' element was found to be located in the region where transcription stopped and showed features reminiscent of some termination sites found in procaryotes. A 5' element was positioned between the P1 and P2 (C. Asselin, A. Nepveu, and K. B. Marcu, Oncogene 4:549-558, 1989). Removal of the more 3' binding site abolished the transcriptional block.

v-Abl activates c-myc transcription through the E2F site.

The v-abl oncogene of Abelson murine leukemia virus encodes a deregulated form of the cellular nonreceptor tyrosine kinase. v-Abl activates c-myc transcription, and c-Myc is an essential downstream component in the v-Abl transformation program. To explore the mechanism by which v-Abl activates c-myc transcription, a cotransfection assay was developed. We show that transactivation of a c-myc promoter by v-Abl requires the SH1 (tyrosine kinase) and SH2 domains of v-Abl; the C-terminal domains are not required for transactivation. The assay also identified the E2F site in the c-myc promoter as a v-Abl-responsive element. In addition, multimerized E2F sites were shown to be sufficient to confer v-Abl-dependent activation on a minimal promoter. This is the first identification of a v-Abl response element for transcriptional activation. v-Abl tyrosine kinase-dependent changes in proteins binding the c-myc E2F site were also demonstrated, including induction of a complex containing DP1, p107, cyclin A, and cdk2. Identification of v-Abl-dependent changes in E2F-binding proteins provides an important link between v-Abl, transcription, cell cycle regulation, and control of cellular growth.

Deregulation of the c-myc oncogene in virus-induced thymic lymphomas of AKR/J mice.

A high frequency (greater than or equal to 65%) of thymomas induced by mink cell focus-forming virus 69L1 in AKR/J mice contain proviral integrations which are clustered 0.7-kilobase upstream of the c-myc oncogene predominantly in the opposite transcriptional orientation. Blot hybridization experiments showed that on the average there was only a twofold elevation of steady-state c-myc RNA in the thymomas as compared with levels in normal AKR/J thymocytes. Such an increase would not appear to be sufficient as a mechanism of oncogene activation in this system. In contrast, S1 nuclease analysis of transcripts initiated from the two known c-myc promoters indicated a strong shift in promoter usage in virtually all thymomas tested. In normal thymus the ratio of transcripts initiated from the proximal promoter P1 to the distal promoter P2 was 0.2 to 0.3. In contrast, most of the thymomas tested (18 of 23) showed an average P1/P2 ratio of 1.2 regardless of whether or not proviral integrations could be detected within a 21-kilobase EcoRI fragment containing the three c-myc exons. We conclude that alterations in P1/P2 ratios are good indicators of c-myc deregulation in thymic lymphomas.

Molecular determinants of NF-kappaB-inducing kinase action.

NF-kappaB corresponds to an inducible eukaryotic transcription factor complex that is negatively regulated in resting cells by its physical assembly with a family of cytoplasmic ankyrin-rich inhibitors termed IkappaB. Stimulation of cells with various proinflammatory cytokines, including tumor necrosis factor alpha (TNF-alpha), induces nuclear NF-kappaB expression. TNF-alpha signaling involves the recruitment of at least three proteins (TRADD, RIP, and TRAF2) to the type 1 TNF-alpha receptor tail, leading to the sequential activation of the downstream NF-kappaB-inducing kinase (NIK) and IkappaB-specific kinases (IKKalpha and IKKbeta). When activated, IKKalpha and IKKbeta directly phosphorylate the two N-terminal regulatory serines within IkappaB alpha, triggering ubiquitination and rapid degradation of this inhibitor in the 26S proteasome. This process liberates the NF-kappaB complex, allowing it to translocate to the nucleus. In studies of NIK, we found that Thr-559 located within the activation loop of its kinase domain regulates NIK action. Alanine substitution of Thr-559 but not other serine or threonine residues within the activation loop abolishes its activity and its ability to phosphorylate and activate IKKalpha. Such a NIK-T559A mutant also dominantly interferes with TNF-alpha induction of NF-kappaB. We also found that ectopically expressed NIK both spontaneously forms oligomers and displays a high level of constitutive activity. Analysis of a series of NIK deletion mutants indicates that multiple subregions of the kinase participate in the formation of these NIK-NIK oligomers. NIK also physically assembles with downstream IKKalpha; however, this interaction is mediated through a discrete C-terminal domain within NIK located between amino acids 735 and 947. When expressed alone, this C-terminal NIK fragment functions as a potent inhibitor of TNF-alpha-mediated induction of NF-kappaB and alone is sufficient to disrupt the physical association of NIK and IKKalpha. Together, these findings provide new insights into the molecular basis for TNF-alpha signaling, suggesting an important role for heterotypic and possibly homotypic interactions of NIK in this response.

Mechanism of c-myc regulation by c-Myb in different cell lineages.

Activation of the murine c-myc promoter by murine c-Myb protein was examined in several cell lines by using a transient expression system in which Myb expression vectors activate the c-myc promoter linked to a chloramphenicol acetyltransferase reporter gene or a genomic beta-globin gene. S1 nuclease protection analyses confirmed that the induction of c-myc by c-Myb was transcriptional and affected both P1 and P2 start sites in a murine T-cell line, EL4, and a myelomonocytic line, WEHI-3. Mutational analyses of the c-myc promoter revealed that two distinct regions could confer Myb responsiveness in two T-cell lines, a distal site upstream of P1 and a proximal site within the first noncoding exon. In contrast, only the proximal site was required for other cell lineages examined. Five separate Myb-binding sites were located in this proximal site and found to be important for c-Myb trans activation. DNA binding was necessary for c-myc activation, as shown by the loss of function associated with mutation of Myb's DNA-binding domain and by trans-dominant repressor activity of the DNA binding, trans-activation-defective mutant. The involvement of additional protein factors was addressed by inhibiting protein synthesis with cycloheximide in a conditional expression system in which the activity of presynthesized Myb was under the control of estrogen. These experiments indicate that de novo synthesis of additional proteins was not necessary for c-myc trans activation. Together these data reveal two cell lineage-dependent pathways by which c-Myb regulates c-myc; however, both pathways are mechanistically indistinguishable in that direct DNA binding by Myb is required for activating c-myc whereas neither de novo protein synthesis nor other labile proteins are necessary.

Human T-cell leukemia virus type 1 Tax induction of NF-kappaB involves activation of the IkappaB kinase alpha (IKKalpha) and IKKbeta cellular kinases.

Tax corresponds to a 40-kDa transforming protein from the pathogenic retrovirus human T-cell leukemia virus type 1 (HTLV-1) that activates nuclear expression of the NF-kappaB/Rel family of transcription factors by an unknown mechanism. Tax expression promotes N-terminal phosphorylation and degradation of IkappaB alpha, a principal cytoplasmic inhibitor of NF-kappaB. Our studies now demonstrate that HTLV-1 Tax activates the recently identified cellular kinases IkappaB kinase alpha (IKKalpha) and IKKbeta, which normally phosphorylate IkappaB alpha on both of its N-terminal regulatory serines in response to tumor necrosis factor alpha (TNF-alpha) and interleukin-1 (IL-1) stimulation. In contrast, a mutant of Tax termed M22, which does not induce NF-kappaB, fails to activate either IKKalpha or IKKbeta. Furthermore, endogenous IKK enzymatic activity was significantly elevated in HTLV-1-infected and Tax-expressing T-cell lines. Transfection of kinase-deficient mutants of IKKalpha and IKKbeta into either human Jurkat T or 293 cells also inhibits NF-kappaB-dependent reporter gene expression induced by Tax. Similarly, a kinase-deficient mutant of NIK (NF-kappaB-inducing kinase), which represents an upstream kinase in the TNF-alpha and IL-1 signaling pathways leading to IKKalpha and IKKbeta activation, blocks Tax induction of NF-kappaB. However, plasma membrane-proximal elements in these proinflammatory cytokine pathways are apparently not involved since dominant negative mutants of the TRAF2 and TRAF6 adaptors, which effectively block signaling through the cytoplasmic tails of the TNF-alpha and IL-1 receptors, respectively, do not inhibit Tax induction of NF-kappaB. Together, these studies demonstrate that HTLV-1 Tax exploits a distal part of the proinflammatory cytokine signaling cascade leading to induction of NF-kappaB. The pathological alteration of this cytokine pathway leading to NF-kappaB activation by Tax may play a central role in HTLV-1-mediated transformation of human T cells, clinically manifested as the adult T-cell leukemia.

Molecular requirements for transcriptional initiation of the murine c-myc gene.

We have identified sequences in the 5' flanking region of the murine c-myc gene's P1 and P2 transcription initiation sites which form specific complexes with nuclear factors of murine and human origin and are also required for normal P1 and P2 usage. Four nuclear factor binding sites were identified within 400 bp 5' of P1 (5'Mf, 5'Mg1, 5'Mg2, and 5'Mg3) and two others within 100 bp 5' of P2 (ME1a1 and ME1a2). The Sp1 transcription factor bound to 5'Mg1 and 5'Mg3 with high affinity and with low affinity to 5'Mg2, ME1a1 and ME1a2 which also bound with high affinity to other factors in crude nuclear extracts. Deletion mutagenesis of sequences 5' of the P1 initiation site revealed that 109 bp encompassing 5'Mg3 and a TATA sequence were sufficient for P1 usage. The ME1a1 binding site 5' of P2 was necessary for maximal P2 activity and the loss of this sequence resulted in enhanced P1 usage. These findings demonstrate that the P1 and P2 initiation sites are independently regulated and that the ME1a1 binding site plays a central role in the normal usage of the c-myc promoters.

Two transient increases in c-myc gene expression during neuroectodermal differentiation of mouse embryonal carcinoma cells.

Mouse embryonal carcinoma (EC) cells of the P19 line can be induced to differentiate into neurons, astrocytes and fibroblasts by exposure to retinoic acid (RA), whereas treatment of the EC cells with dimethyl sulfoxide (DMSO) leads to differentiation into mesodermal tissues, including cardiac and skeletal muscle. In RA- but not DMSO-treated cultures, the level of c-myc mRNA underwent two transient increases. The concentration of c-myc mRNA in RA-treated cultures increased 3-fold after 3 h in the presence of the drug, returned to normal by 9 h, increased again 5-fold from 48 to 96 h, and finally decreased below pre-treatment values by 144 h. Increased levels of c-myc protein were observed at the times of elevated c-myc mRNA. Nuclear run-on assays of the c-myc gene and measurements of c-myc mRNA stability indicated that both transcriptional and post-transcriptional mechanisms contribute to the modulated expression of the c-myc gene. The two transient increases in c-myc expression suggest a role for the c-myc protein in the differentiation of cells along neuroectodermal lineages.

Multiple negative elements upstream of the murine c-myc gene share nuclear factor binding sites with SV40 and polyoma enhancers.

We previously identified a 716 bp DNA segment, 424 to 1140 bp 5' of the murine c-myc gene, which exhibited the properties of a transcriptional 'dehancer' because it negated the effects of the SV40 enhancer (Remmers et al., 1986). Here, we show that this 716 bp 'dehancer region' is composed of multiple negative elements each of which functions at a distance of > 1 Kb to inhibit the SV40 enhancer. One of these negative elements displays lymphoid cell specificity. HeLa cell nuclear factor binding sites are found within these negative effector sequences. One of these binding sites is related to that of the Py EF-C transcription factor which recognizes a short dyad symmetry element within the polyoma enhancer. Interestingly, a dimer of the Py EF-C recognition sequence exhibited a strong 'dehancer effect' in the pSV2CAT expression vector. These negative elements may function by interfering with transcription factors required for SV40 enhancer activity.

Tissue-specific post-transcriptional regulation of c-myc expression in normal and H-2K/human c-myc transgenic mice.

We show that the steady-state levels of c-myc mRNAs vary considerably in different organs of normal adult mice, maximal expression being observed in lymphoid organs and minimal expression in liver and brain. Nuclear run-on analysis of c-myc gene transcription in adult liver and spleen reveals that the difference in c-myc gene expression in these two organs is due to differential post-transcriptional control. Moreover, these nuclear run-on assays indicate that no premature termination of c-myc gene transcription takes place in the nuclei of the three adult tissues analysed. In fetal liver development, we observe a decrease in c-myc mRNA, but this is not due to changes in transcriptional activity implicating post-transcriptional regulatory mechanisms. Our studies of c-myc gene expression in organs of H-2K/myc transgenic mice, harboring an H-2K promoter driven human c-myc gene, confirm that the in vivo c-myc regulation is mainly post-transcriptional and shows that sequences shared by the murine and human c-myc proto-oncogenes are involved in this control.

Elevated expression of an exogenous c-myc gene is insufficient for transformation and tumorigenic conversion of established fibroblasts.

Two established rat fibroblast lines, differing only by their number of generations in culture, show dramatically different responses to the elevated c-myc expression delivered by an efficient murine c-myc retrovirus vector. Thus, a late passage (60 generation) FR3T3 line acquires a transformed and tumorigenic phenotype upon introduction of this activated c-myc gene as indicated by its altered morphology, high efficiency of focus formation, soft agar clonability, saturation density in monolayer culture, and short latency of tumorigenicity in syngeneic hosts. Remarkably, none of these characteristics, except for an increased refractility in monolayers and an epidermal growth factor (EGF)-dependent agar clonability, were observed in a variety of early passage (10 generation) FR3T3 c-myc clones. BALB/c A31 fibroblasts transfected with this c-myc retroviral vector behaved essentially the same as the FR3T3 early line except for their inability to grow in suspension in response to EGF. However, transformation and tumorigenic conversion of each of these three fibroblast lines was achieved by an activated ras oncogene. Hence, elevated c-myc expression is insufficient for transformation of established fibroblasts but depends upon other acquired cooperating functions which are not necessary for ras induced transformation. We also demonstrate that endogenous c-myc expression remains unaffected even in clones expressing a 100-fold excess of exogenous c-myc RNAs demonstrating that c-myc autoregulation is not operative in these cells.