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K Calame

Publications and source records attributed to K Calame.

At least 19 recordsLinked to original sources
PubMed1998

Transcription of a minimal promoter from the NF-IL6 gene is regulated by CREB/ATF and SP1 proteins in U937 promonocytic cells.

NF-IL6 is an important transcriptional regulator of genes induced in activated monocytes/macrophages, and NF-IL6 is the only CCAAT/enhancer-binding protein (C/EBP) family member whose steady-state mRNA levels increase upon activation of monocytes (1). We show that increased transcription of the NF-IL6 gene is responsible, at least in part, for induction of NF-IL6 mRNA following activation of U937 promonocytic cells. We have identified a 104-bp minimal promoter region of the NF-IL6 gene that is sufficient for basal and activation-dependent induction of transcription in U937 cells. This region contains binding sites for the cAMP response element-binding protein/activation transcription factor (CREB/ATF) and Sp1 families of transcription factors. Each site is functionally important and contributes independently to transcription of the NF-IL6 gene in U937 cells.

Activating Transcription Factors↗
PubMed1998

Evidence that immunoglobulin VH-DJ recombination does not require germ line transcription of the recombining variable gene segment.

The importance of V(D)J recombination for generating diversity in the immune system is well established, but the mechanisms which regulate V(D)J recombination are still poorly understood. Although transcription of unrearranged (germ line) immunoglobulin and T-cell receptor gene segments often precedes V(D)J recombination and has been implicated in its control, the actual role of germ line transcripts in V(D)J recombination is not known. We used a sensitive reverse transcription-PCR assay to study immunoglobulin VH germ line transcripts in proB lines from RAG-deficient mice. All 10 VH families analyzed were germ line transcribed, and germ line transcription was found in all of the cell lines examined, indicating that active chromatin was present in the VH region. However, not all VH families were germ line transcribed in every cell line, and there was a surprising lack of uniformity in the number and family distribution of germ line VH transcripts in individual lines. When V(D)J recombination was activated by restoration of RAG activity, recombinational activity of endogenous VH genes for which germ line transcription was observed could be compared with those of genes for which it was not observed. This analysis revealed multiple examples of endogenous VH gene segments which were rearranged in cells where their germ line transcription was not detectable prior to RAG expression. Thus, our data provide strong support for the idea that V-(D)J recombination does not require germ line transcription of the recombining variable gene segment.

Animals↗
PubMed1998

Transcriptional regulation during B cell development.

Information is increasingly available concerning the molecular events that occur during primary and antigen-dependent stages of B cell development. In this review the roles of transcription factors and coactivators are discussed with respect to changes in expression patterns of various genes during B cell development. Transcriptional regulation is also discussed in the context of developmentally regulated immunoglobulin gene V(D)J recombination, somatic hypermutation, and isotype switch recombination.

Animals↗
PubMed1997

Repression of c-myc transcription by Blimp-1, an inducer of terminal B cell differentiation.

Transcription of c-myc in plasma cells, which are terminally differentiated B cells, is repressed by plasmacytoma repressor factor. This factor was identified as Blimp-1, known for its ability to induce B cell differentiation. Blimp-1 repressed c-myc promoter activity in a binding site-dependent manner. Treatment of BCL1 lymphoma cells with interleukin-2 (IL-2) plus IL-5 induced Blimp-1 and caused a subsequent decline in c-Myc protein. Ectopic expression of Blimp-1 in Abelson-transformed precursor B cells repressed endogenous c-Myc and caused apoptosis; Blimp-1-induced death was partially overcome by ectopic expression of c-Myc. Thus, repression of c-myc is a component of the Blimp-1 program of terminal B cell differentiation.

Animals↗
PubMed1997

The ZiN/POZ domain of ZF5 is required for both transcriptional activation and repression.

ZF5 is a ubiquitously expressed protein originally identified by its ability to bind and repress the murine c-myc promoter. It contains five C-terminal zinc fingers and a conserved N-terminal ZiN/POZ domain. This motif, found in a growing number of zinc finger proteins, can inhibit DNA binding and mediate dimerization [Bardwell, V.J. and Treisman,R. (1994) Genes Dev., 8,1664-1677]. In the current study, a cyclic amplification and selection of targets (CAST) protocol detected preferred ZF5 binding sites which are highly GC-rich. Binding to these sites by ZF5 depended upon the zinc fingers and was enhanced when the ZiN/POZ domain was removed. Using transient cotransfection assays, ZF5 was shown to activate the HIV-1 LTR and repress the beta-actin promoter. The ZiN/POZ domain was shown to mediate ZF5-dependent transcriptional activation and repression. From these data, we conclude that ZF5 can both activate and repress in the context of different natural promoters and that its ZiN/POZ domain can affect two functions; DNA binding and transcriptional modulation.

3T3 Cells↗
PubMed1997

Induction of c-myc transcription by the v-Abl tyrosine kinase requires Ras, Raf1, and cyclin-dependent kinases.

v-Abl is an oncogenic form of the c-Abl nonreceptor tyrosine kinase. v-Abl induces transcription of c-myc, and c-Myc function is a necessary but not sufficient component of the v-Abl transformation program. Previously we showed that the E2F site in the c-myc promoter is a v-Abl response element and that v-Abl appears to induce c-myc by initiating a phosphorylation cascade that ultimately activates E2F-binding proteins. In this work we have investigated the signaling pathway between the v-Abl tyrosine kinase and activated E2F proteins. We show that the Ras GTPase and Raf1 serine/threonine kinase are required in this pathway. However, in contrast to other aspects of v-Abl signaling, induction of c-myc transcription is independent of the Rac GTPase. Our results also establish a requirement for activated cyclin-dependent kinases (cdks), as v-Abl-dependent induction of c-myc transcription is blocked by cdk inhibitor p21 and induction of c-myc is accompanied by activation of cdk2 and cdk4. Finally, we show that v-Abl-dependent induction of c-myc is accompanied by hyperphosphorylation of pRb, p107, and p130. On the basis of these data, we propose a model for the signaling path from v-Abl to c-myc.

Animals↗
PubMed1997

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↗
PubMed1996

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↗
PubMed1996

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↗
PubMed1995

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↗
PubMed1995

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↗
PubMed1995

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↗
PubMed1995

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↗
PubMed1995

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↗
PubMed1994

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↗