Positive and negative regulation of c-Myc transcription.
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Animals↗
Biomedical subjects
K Calame
Publications and source records attributed to K Calame.
The absence of the transcription activator TFE3 impairs activation of B cells in vivo.
TFE3 is a ubiquitously expressed member of the TFE3/mi family of basic helix loop helix zipper transcription factors. TFE3 binds to muE3 sites located in the immunoglobulin heavy-chain (IgH) intronic enhancer, heavy-chain variable region promoters, the Ig kappa intronic enhancer, and regulatory sites in other genes. To understand the role of TFE3 in Ig expression and lymphoid development, we used embryonic stem (ES) cell-mediated gene targeting and RAG2-/- blastocyst complementation to generate mice which lack TFE3 in their B and T lymphocytes. TFE3- ES cells fully reconstitute the B- and T-cell compartments, giving rise to normal patterns of IgM+ B220+ B cells and CD4+ and CD8+ T cells. However, TFE3- B cells show several defects consistent with poor B-cell activation. Serum IgM levels are reduced twofold and IgG and IgA isotypes are reduced three- to sixfold in the TFE3- chimeras even though in vitro, the TFE3- splenocytes secrete normal levels of all isotypes in response to lipopolysaccharide activation. Peripheral TFE3- B cells also show reduced surface expression of CD23 and CD24 (heat-stable antigen).
Alleles↗
YY1 and c-Myc associate in vivo in a manner that depends on c-Myc levels.
The c-Myc oncoprotein has previously been shown to associate with transcription regulator YY1 and to inhibit its activity. We show herein that endogenous c-Myc and YY1 associate in vivo and that changes in c-Myc levels, which accompany mitogenic stimulation or differentiation of cultured cells, affect the ratio of free to c-Myc-associated YY1. We have also investigated the mechanism by which association with c-Myc inhibits YY1's ability to regulate transcription. c-Myc does not block binding of YY1 to DNA. However, protein association studies suggest that c-Myc interferes with the ability of YY1 to contact basal transcription proteins TATA-binding protein and TFIIB.
3T3 Cells↗
A 125 bp region of the Ig VH1 promoter is sufficient to confer lymphocyte-specific expression in transgenic mice.
Ig variable region (VH) promoters are active only in B cells and extinction experiments suggest that they are negatively regulated in non-B lineage cells. In contrast to the multiple transcription factor binding sites which occur in Ig enhancers, only a few functionally important transcription factor binding sites have been identified in VH promoters. In this study, we have used transgenic animals to test the functional importance of a 5' portion of the VH1 promoter which is known to contain a matrix attachment region as well as binding sites for the negative regulator NF-microNR and Bright, a protein complex which is induced upon stimulation of B cells with IL-5 and antigen. Our results show that none of these regions is required for VH1 promoter activity in the context of a rearranged micro gene. In fact, a truncated promoter extending only 125 bp upstream of the transcription initiation site was sufficient to confer strong expression in lymphocytes with negligible expression in any other tissue. These results define the smallest known region of a VH promoter which is capable of lymphoid-specific activity and establish the simplicity of the VH1 promoter element.
Animals↗
Ig/EBP (C/EBP gamma) is a transdominant negative inhibitor of C/EBP family transcriptional activators.
Analysis of cDNA and genomic clones shows that the murine Ig/EBP (C/EBP gamma) gene encodes a small protein with a predicted molecular weight of 16.4 kDa which contains C/EBP family basic and leucine zipper domains but lacks the transcriptional activation domains present in C/EBP (C/EBP alpha) and NF-IL6 (C/EBP beta). In transfection assays Ig/EBP is neither an activator nor a repressor of transcription; however, Ig/EBP inhibits the transcriptional ability of NF-IL6 (C/EBP beta) and C/EBP (C/EBP alpha), acting as a transdominant negative regulator. Thus Ig/EBP resembles LIP, another negative regulator of the C/EBP family, in both structure and transcriptional activity. Of the three known C/EBP family inhibitors, Ig/EBP, LIP and CHOP-10, only Ig/EBP is ubiquitously expressed. Therefore, Ig/EBP may act as a general buffer for C/EBP activators in many cell types.
Animals↗
TFE3 contains two activation domains, one acidic and the other proline-rich, that synergistically activate transcription.
TFE3 is a basic-helix-loop-helix-zipper (bHLHZIP) domain-containing protein that binds mu E3 sites in regulatory elements in the immunoglobulin heavy chain gene. The protein is a transcriptional activator that is expressed in vivo as two alternately spliced isoforms with different activating properties: TFE3L contains an N-terminal acidic activation domain; TFE3S lacks this activation domain and is a dominant negative inhibitor of TFE3L. We show that TFE3L and TFE3S contain a second, C-terminal activation domain rich in proline residues. This pro-rich activation domain has activity in a Gal4 fusion assay comparable to the N-terminal acidic activation domain present in TFE3L. The TFE3 pro-rich activation domain contains regions of strong homology with the related proteins microphthalmia and TFEB, suggesting that these regions are important for function. Using two different assays, we show that the N- and C-terminal activation domains of TFE3 act synergistically. This synergism explains in part the ability of TFE3S to act as a dominant negative. Our domain analysis of TFE3 is incorporated into a general structural model for the TFE3 protein that predicts that the activation domains of TFE3 will be widely separated in space.
3T3 Cells↗
Polyamines alter sequence-specific DNA-protein interactions.
The polyamines are abundant biogenic cations implicated in many biological processes. Despite a plethora of evidence on polyamine-induced DNA conformational changes, no thorough study of their effects on the activities of sequence-specific DNA binding proteins has been performed. We describe the in vitro effects of polyamines on the activities of purified, representative DNA-binding proteins, and on complex protein mixtures. Polyamines at physiological concentrations enhance the binding of several proteins to DNA (e.g. USF, TFE3, Ig/EBP, NF-IL6, YY1 and ICP-4, a herpes simplex virus gene regulator), but inhibit others (e.g. Oct-1). The degree of enhancement correlates with cationic charge; divalent putrescine is ineffective whereas tetravalent spermine is more potent than trivalent spermidine. Polyamine effects on USF and ICP-4 result from increased rate of complex formation rather than a decreased rate of dissociation. DNAse I footprint analysis indicated that polyamines do not alter DNA-protein contacts. Polyamines also facilitate formation of complexes involving binding of more than one protein on a DNA fragment.
Base Sequence↗
Association with c-Myc: an alternated mechanism for c-Myc function.
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DNA-Binding Proteins↗
Genetic analyses of tattered, an X-linked dominant, developmental mouse mutation.
Tattered (Td) is an X-linked dominant mouse mutation that causes prenatal lethality in affected males. To map the locus, we analyzed 199 normal male and affected female progeny from a backcross of Td and Mus castaneus. Pedigree analysis of these animals suggests a gene order of cen-DXWas70-(Td, DXMit26, Gata1, Tcfe3)-(Cybb, Otc)-tel, where Tcfe3 is a transcription factor homologous to a gene involved in the murine microphthalmia (mi) mutation [Hodgkinson et al. Cell 74, 395-404, 1993]. To evaluate Tcfe3 as a candidate for Td, heterozygous tattered females were crossed to xid males to obtain females in which > 95% of B cells expressed genes solely from the Td X Chromosome (Chr). Fluorescent activated cell sorting (FACS) analysis and Western blotting of isolated splenocytes from Td/xid double heterozygotes rule out Tcfe3 as a likely candidate for the Td mutation.
Animals↗
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.
Abelson murine leukemia virus↗
An analysis of genes regulated by the multi-functional transcriptional regulator Yin Yang-1.
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Amino Acid Sequence↗
The C/EBP family of proteins distorts DNA upon binding but does not introduce a large directed bend.
The DNA-bending properties of several C/EBP family proteins, bound to two different sites, have been determined using circular permutation and phasing analyses. All the proteins examined by circular permutation analysis induced a significant distortion in the DNA (40-66 degrees), and this distortion was of the same magnitude at both C/EBP sites. However, phasing analysis revealed that the size of the directed bend induced in the DNA by C/EBP proteins was only 1-4 degrees. Both the magnitude and orientation of the directed bend were affected by the specific sequence of the DNA-binding site. All proteins induced a directed bend of similar magnitude and orientation (toward the minor groove) when bound to the C/EBP site derived from the IgH enhancer, but the induced bend was smaller when the protein was bound to the C/EBP site derived from the VH1 promoter. The study also included a heterodimer between Ig/EBP and ATF4. Similar to C/EBP protein homodimers, this heterodimer induces a small directed bend of 4 degrees oriented toward the minor groove when bound to the enhancer-derived C/EBP site.
Base Sequence↗
The basic helix-loop-helix-zipper domain of TFE3 mediates enhancer-promoter interaction.
Binding sites for three families of sequence-specific DNA-binding proteins, microE3, C/EBP, and OCT, are found in both the promoters and the intronic enhancer of the immunoglobulin heavy-chain gene. We have used a cotransfection system to investigate how proteins binding these sites may participate in enhancer-promoter interactions. Basic helix-loop-helix-zipper (BHLHZIP) proteins TFE3 and TFEB activate from a distance in this assay, but the basic zipper (BZIP) protein NF-IL6 and endogenous OCT-binding proteins do not. Our results suggest that remotely bound TFE3 is recruited to the initiation site by association with proximally bound TFE3; this interaction is mediated by the BHLHZIP domain and not by activation domains of TFE3. The BZIP domain of Ig/EBP lacks this activity, revealing an important functional difference between these structurally related dimerization domains. We also show that TFE3 can exist as a tetramer in solution and that tetramerization is determined by the HLHZIP domain. These data support a model in which protein-protein interactions between proximally and remotely bound TFE3 recruit TFE3 to the initiation site for activation. The IgH gene is the first example of a cellular gene in which proximal and distal binding sites are found for a protein capable of mediating enhancer-promoter interaction.
3T3 Cells↗
Inhibition of transcriptional regulator Yin-Yang-1 by association with c-Myc.
Yin-Yang-1 (YY1) regulates the transcription of many genes, including the oncogenes c-fos and c-myc. Depending on the context, YY1 acts as a transcriptional repressor, a transcriptional activator, or a transcriptional initiator. The yeast two-hybrid system was used to screen a human complementary DNA (cDNA) library for proteins that associate with YY1, and a c-myc cDNA was isolated. Affinity chromatography confirmed that YY1 associates with c-Myc but not with Max. In cotransfections, c-Myc inhibits both the repressor and the activator functions of YY1, which suggests that one way c-Myc acts is by modulating the activity of YY1.
3T3 Cells↗
Transcriptional repressor ZF5 identifies a new conserved domain in zinc finger proteins.
We have cloned a cDNA encoding a new murine C2H2 zinc finger protein, ZF5. The 51.3 kD protein contains five GL1-Kruppel type zinc fingers at the C-terminus. At its N-terminus, ZF5 has a 41 amino acid region which was found to be homologous to the N-termini of several other zinc finger proteins. This region defines a new motif within zinc finger proteins which we have named the Zinc finger N-terminal (ZiN) domain. ZF5 binds to two sites in the c-myc promoter and to the -50 bp site of the herpes simplex thymidine kinase promoter. ZF5 is a transcriptional repressor and its repression domain is located N-terminal to the zinc finger domains. A single 4 kb ZF5 mRNA is expressed widely.
Amino Acid Sequence↗
Yin-yang 1 activates the c-myc promoter.
Previous studies on the murine c-myc promoter demonstrated that a ubiquitously present protein, common factor 1 (CF1), bound at two sites located -260 and -390 bp from the P1 transcription start site. CF1 has been purified to near homogeneity and shown to be identical to the zinc finger protein Yin-yang 1 (YY1) as judged by similarity of molecular weight and other biochemical properties, immunological cross-reactivity, and the ability of recombinant YY1 to bind to CF1 sites. In cotransfection experiments, YY1 is a strong activator of transcription from c-myc promoter-based reporters. Furthermore, in murine erythroleukemia cells, overexpressed YY1 causes increased levels of c-myc mRNA initiated from both major transcription initiation sites of the endogenous c-myc gene.
3T3 Cells↗
The C/EBP family of transcriptional activators is functionally important for Ig VH promoter activity in vivo and in vitro.
We have examined the functional importance of binding sites for C/EBP family members (E sites), in two Ig VH promoters: VH1, a member of the S107 family, and BCL1, a member of the J558 family. Mutation of the E site in the VH1 promoter diminishes transcription in vivo to 59% of wild-type and transcription from the BCL1 promoter in vitro is inhibited to an average of 39% of wild-type by competition with E site oligonucleotides. Purified E site binding proteins from plasmacytoma cells stimulated BCL1 transcription in vitro 2.3-fold. Although five C/EBP family proteins are known which bind the E site, antibody ablation of DNA:protein complexes resolved by electrophoretic mobility shift assays showed that Ig/EBP-1 is the only E site binding protein detectable in early B cell lines; more mature B cells contain Ig/EBP-1 and NF-IL6. We also show by antibody-depletion that Ig/EBP-1 activates the BCL1 promoter in vitro.
Animals↗
mTFE3, an X-linked transcriptional activator containing basic helix-loop-helix and zipper domains, utilizes the zipper to stabilize both DNA binding and multimerization.
Southwestern (DNA-protein) screening of a murine L-cell cDNA library by using a probe for the microE3 site in the immunoglobulin heavy-chain enhancer yielded a clone, mTFE3, which is a member of the subset of basic helix-loop-helix (BHLH) proteins that also contain a leucine zipper (ZIP). Since the individual contribution of these domains is not well understood for proteins which contain them both, mutational analyses were performed to assess the functional roles of the HLH and ZIP regions for DNA binding and multimerization. The HLH region is stringently required for DNA binding but not for multimerization. The ZIP region is not stringently required for binding or multimerization, but stabilizes both multimer formation and DNA binding. A high degree of conservation at both the amino acid and nucleotide levels between the human transcription factor TFE3 and mTFE3 suggests that mTFE3 is the murine homolog of human TFE3. By using fluorescent in situ hybridization, mTFE3 was mapped to mouse chromosome X in band A2, which is just below the centromere. We show that in addition to the immunoglobulin heavy-chain microE3 site, mTFE3 binds to transcriptional elements important for lymphoid-specific, muscle-specific, and ubiquitously expressed genes. Binding of mTFE3 to DNA induces DNA bending.
Amino Acid Sequence↗