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K B Marcu

Publications and source records attributed to K B Marcu.

At least 37 records · Page 2Linked to original sources
PubMed1995

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.

Base Sequence↗
PubMed1995

CHUK, a new member of the helix-loop-helix and leucine zipper families of interacting proteins, contains a serine-threonine kinase catalytic domain.

We have identified a new member of the helix-loop-helix (H-L-H) and leucine zipper gene families via a reverse transcriptase-polymerase chain reaction based strategy. This new gene, CHUK (conserved helix-loop-helix ubiquitous kinase), may represent the founding member of a new class of interacting chimeric proteins. The nucleotide sequence of a near full-length murine CHUK cDNA clone revealed an encoded polypeptide specifying: a carboxyl-terminal H-L-H domain, an amino terminal serine-threonine kinase catalytic domain, and a leucine zipper-like amphipathic alpha-helix juxtaposed in between the H-L-H and kinase domains. CHUK is highly conserved in evolution and ubiquitously expressed in diverse types of established cell lines, whereas it is differentially expressed in normal murine tissues. The structural features of the CHUK polypeptide suggest that its putative kinase activity may be targetted to H-L-H and/or leucine zipper transcription factors. Alternatively, the dual amphipathic a helices may serve to control its intrinsic kinase activity by interactions with other cellular factors. CHUK may provide new insights into the regulated transmission of cytoplasmic signals to specific nuclear factors manifesting rapid alterations in patterns of cellular gene expression.

Amino Acid Sequence↗
PubMed1994

MAZ-dependent termination between closely spaced human complement genes.

The zinc finger protein MAZ, originally identified as a factor that binds to the c-myc P2 promoter, is associated with transcriptional termination. As shown in these studies, a termination sequence between the closely spaced human complement genes C2 and Factor B contains a protein binding site which interacts with three different proteins in vitro. Binding of one of these factors, MAZ, correlates with activity of the C2 termination sequence in vivo. Cloned MAZ was used to obtain a consensus binding site, G5AG5. This allowed identification of new sites, between the closely spaced human genes g11 and C4 and within an intron of the mouse IgM-D gene, where termination is known to occur and regulate the expression of IgD. The g11 and IgM MAZ sites lie within sequences that have activity in a termination assay and, furthermore, mutation of C2 or g11 MAZ sites severely reduces termination activity. MAZ bends DNA, and inherently bent DNA is highly active as a terminator, suggesting that MAZ-induced bending is important for C2 and g11 termination. We propose that MAZ sites exist in promoters which require protection against transcriptional interference, such as those of closely spaced genes, to cause efficient termination. The MAZ consensus sequence will facilitate the identification of further sites.

Base Sequence↗
PubMed1994

PANG, a gene encoding a neuronal glycoprotein, is ectopically activated by intracisternal A-type particle long terminal repeats in murine plasmacytomas.

Plasmacytomagenesis provides a murine model to decipher progressive genetic events culminating in a B-cell neoplasia. Activation of the c-myc protooncogene by chromosomal translocation is considered an initiating event. Intracisternal A-type particles (IAPs) are defective retroviral-like structures present in the endoplasmic reticulum of plasmacytomas (PCTs). IAP proviral insertions have been documented to engender negative or positive effects on the expression of nearby cellular genes. We have isolated a gene, PANG (plasmacytoma-associated neuronal glycoprotein), that is ectopically transcribed in a number of PCTs due to IAP long terminal repeat (LTR) activation. A full-length PANG cDNA was isolated from an MPC-11 plasma cell tumor cDNA library and encodes a polypeptide of about 113 kDa with six immunoglobulin C2-like and four type III fibronectin-like domains. PANG bears a striking resemblance to axonal glycoproteins TAG-1 and F11 known to function in neuronal outgrowth. An extensive survey revealed a predominant 3.6-kb PANG transcript in 60% (30 of 50) of PCTs as well as unique smaller and larger species. All other normal and transformed lymphoid and nonlymphoid cell lines and normal tissues were negative for PANG expression except for the brain, wherein unique 4.0- and 6.1-kb transcripts were detected. Reverse transcriptase PCR analysis revealed IAP LTR fusion to PANG mRNAs in five PCTs and in a neuroblastoma line. The 5' end of a mouse brain PANG cDNA was identical to the MPC-11 PANG transcript except for the precise replacement of its 5' LTR sequence.

Amino Acid Sequence↗
PubMed1994

The Epstein-Barr virus BRLF1 gene product transactivates the murine and human c-myc promoters.

A common feature of the Epstein-Barr virus (EBV)-associated malignancy, Burkitt's lymphoma, is chromosomal translocation affecting the c-myc oncogene. We report here that an EBV immediate-early (IE) protein, BRLF1 (R), transactivates the murine and human c-myc promoters. The R transactivator enhances expression from transiently transfected murine and human c-myc promoters as determined both at the CAT reporter and at the mRNA level. Transactivation of the human c-myc promoter by R occurs in several different cell lines, and this effect is reporter-gene independent. Both the P1 and P2 c-myc promoters can be activated by the EBV R IE protein, although the R-induced transactivation of P1 is greater than P2. The portion of the human c-myc promoter from -228 to -63 (relative to the P1 mRNA start site) is necessary, but not sufficient, for transactivation by R in the Louckes B-cell line. Binding of the R protein directly to the c-myc promoter could not be demonstrated, suggesting that the effect of R on c-myc activity occurs by an indirect mechanism. The ability of an EBV protein to activate c-myc expression is likely to facilitate productive viral infection and is also potentially relevant in the genesis of EBV-associated lymphomas.

Animals↗
PubMed1993

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.

Animals↗
PubMed1992

MAZ, a zinc finger protein, binds to c-MYC and C2 gene sequences regulating transcriptional initiation and termination.

ME1a1, a 16-base-pair nuclear factor binding site residing between the c-MYC P1 and P2 transcription initiation sites, is required for P2 activity. A cDNA encoding a 477-amino acid zinc finger protein designated MAZ (MYC-associated zinc finger protein) was cloned from a HeLa lambda gt11 library by screening with a concatamerized ME1a1 binding site probe. In addition to six potential zinc fingers of the Cys2His2 type, MAZ contains an amino-terminal proline-rich domain and several polyalanine tracts. Its mRNA was present in all human tissues tested except for kidney, as a doublet of approximately 2.5 and 2.7 kilobases, along with differentially expressed minor species. MAZ bound specifically to the wild-type ME1a1 sequence but not to a ME1a1 mutant that also failed to yield P2 activity. When expressed as a fusion protein in a pMAL-c vector, MAZ binds with specificity to a GA box sequence (GGGAGGG) found in the c-MYC P2 promoter, to the P2 attenuator region within the gene's first exon, and to a related sequence involved in the transcriptional termination of the C2 gene. MAZ may encode a transcription factor with dual roles in transcription initiation and termination.

Amino Acid Sequence↗
PubMed1992

Properties of B cell stage specific and ubiquitous nuclear factors binding to immunoglobulin heavy chain gene switch regions.

The Ig heavy chain (IgH) constant region (CH) class switch is manifested by DNA deletions which exchange the C mu gene of a functional VDJ-CH rearrangement for a C gamma, C epsilon or C alpha gene. Repetitive sequences (S regions) 5' of each CH gene mediate CH gene switch recombination by an illegitimate mechanism. S mu can be subdivided into S mu 5' (non-repetitive) and S mu 3' (repetitive) components with recombination occurring in either part. Here, we describe the properties of ubiquitous and B cell stage specific S mu binding factors NFS mu-U1 and NFS mu-B1 respectively. U1 only bound to S mu 5' sequences, and B1 to S mu 5', S mu 3' sequences and to other S regions with varying affinities. DMS and OP-Cu footprinting revealed the sequence AAAAAGCATGGCTGA in the U1 site while the B1 S mu 5' site overlapped the 3' end of the U1 binding site and also contained additional 3' flanking S mu repeat motifs (GAGCTGAGATGGGTGGGCT). Binding site competition assays reveal that NFS mu-B1 is either very related or identical to S alpha BP (described by Waters et al., Mol. Cell Biol. 9:5594, 1989) and BSAP (identified by Barberis et al., Genes Devl. 4:849, 1990) which were shown to bind to two sequences upstream of the S alpha repeats and within the promoters of sea urchin histone genes respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

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.

Animals↗
PubMed1990

On the mechanism of non-allelically excluded V alpha-J alpha T cell receptor secondary rearrangements in a murine T cell lymphoma.

We had previously demonstrated that several subclones derived from a CD3+, CD4-/CD8-, TCR-alpha beta+ murine T cell line have undergone secondary V alpha-J alpha rearrangements at the TCR-alpha locus (1). In an effort to examine the molecular mechanism responsible for these V alpha-J alpha replacements, the structures of TCR-alpha cDNA prepared from both the parental and subcloned T cell lines have been determined. Here we report that: 1) the mechanism whereby the secondary rearrangements occur is a precise deletion event that involves germ-line V alpha genes 5' to the preexisting V alpha-J alpha complex joining to J alpha segments 3' of the preexisting complex deleting the region in between, 2) preexisting productive V alpha-J alpha rearrangements of the parental line do not allelically exclude productive and nonproductive secondary rearrangements, 3) both productively rearranged TCR-alpha alleles of the parental cell line can undergo secondary rearrangements, 4) the presence of unrearranged germline V alpha transcripts in the parental line support an "accessibility" model of regulated lymphocyte receptor gene rearrangement. In addition, we present data which suggests that one of the subcloned lines has undergone a third rearrangement of one of its TCR-alpha alleles. One interpretation of these results is that T cells may have the ability to circumvent allelic exclusion at the TCR-alpha locus early in their ontogeny. This could provide T cells with an additional mechanism for generating an Ag receptor repertoire which is not found in B cells.

Alleles↗
PubMed1990

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.

Animals↗
PubMed1989

Molecular requirements for immunoglobulin heavy chain constant region gene switch-recombination revealed with switch-substrate retroviruses.

We have employed a retroviral vector, ZN(Smu/S gamma 2b)tk1, as a means of introducing immunoglobulin heavy chain (IgH) switch (S) region sequences into B cell lines to directly measure their switch-recombinase activities. In an earlier study, we demonstrated that retrovector Smu-S gamma 2b recombination events occurred in two thymidine kinase (tk)-negative murine pre-B cell lines (18-8 and 38B9) upon selection in bromodeoxyuridine (BUdR) media for the loss of an Htk gene inserted in between the vector's Smu and S gamma 2b sequences. Here we have used this assay system to show that the 300-18 murine pre-B cell line possesses a very high level of switch-recombinase activity (greater than 1 event in 2500 cells/generation) while a terminally differentiated, antibody-secreting hybridoma line (A39R 1.1) has no detectable recombinase activity. Both S mu and S gamma 2b segments are required for switch region-mediated deletions. Retrovectors harboring only an Smu segment or an Smu segment and a portion of the murine c-myc gene in place of S gamma 2b sequences were both non-recombinagenic in this assay system. Nucleotide sequence analysis of six retrovector S segment recombinants, recovered from ZN(Smu/S gamma 2b) tk1-infected 18-8 and 39B9 pre-B lines, did not reveal homology at their sites of recombination. We conclude that: (1) S segment repetitive sequences play an essential but indirect role in IgCH gene switch-recombination, which occurs by an illegitimate, non-homologous mechanism; (2) the c-myc gene is not a significant target for switch-recombination; and (3) since endogenous Smu and S gamma 2b rearrangements were not observed in populations and clones of pre-B cells expressing a high level of switch-recombinase activity, multiple factors (presumably contributed in part by the degree of S segment accessibility) in addition to S recombinase activity are required for CH class switching.

Animals↗
PubMed1989

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.

Animals↗
PubMed1989

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.

Animals↗