Alternative splicing and embryonic expression of the Xenopus mad4 bHLH gene.
(17/1416)
The cell proliferative activity of the Myc family of basic helix-loop-helix/leucine zipper (bHLHZip) transcription factors is dependent upon binding to the ubiquitous Max protein. In the absence of heterodimerization with Max, Myc protein is unable to efficiently bind to DNA and activate transcription. Members of the Mad family of transcription factors are thought to modulate the cell proliferative effects of the c-myc proto-oncogene by binding to Max, directly competing with the Myc protein for both heterodimerization and DNA binding. Consistent with a role in down-regulating cell division, the murine mad genes are expressed in embryonic tissues undergoing differentiation, often during or shortly after the down-regulation of myc gene expression. Here, we report the isolation and characterization of the first Xenopus mad family member, Xmad4. Maternal Xmad4 transcripts are present at high levels in the oocyte and in the cleavage stage embryo, but almost disappear by the neurula stage. Zygotic expression of the Xmad4 gene is initiated in the epidermis of the late neurula stage, and shortly thereafter, Xmad4 is transiently detectable in the cement and hatching glands. At later stages, expression is also observed in the developing pronephros and liver. Unlike the murine mad4 gene, we find that multiple Xmad4 splice variants exist in Xenopus and that these variants are differentially expressed in both the embryo and the adult. Despite the demonstrated antagonistic role of Mad proteins in the regulation of Myc activity, we show that the over-expression of Xmad4 in the cleavage-stage embryo has no detectable phenotypic effect, suggesting that Myc function is dispensable during early embryonic development. (+info)
New Myc-interacting proteins: a second Myc network emerges.
(18/1416)
Despite its intensive investigation for almost two decades, c-Myc remains a fascinating and enigmatic subject. A large and compelling body of evidence indicates that c-Myc is a transcription factor with central roles in the regulation of cell proliferation, differentiation, and apoptosis, but its exact function has remained elusive. In this review we survey recent advances in the identification and analysis of c-Myc-binding proteins, which suggest insights into the transcriptional roles of c-Myc but which also extend the existing functional paradigms. The C-terminal domain (CTD) of c-Myc mediates interaction with Max and physiological recognition of DNA target sequences, events needed for all biological actions. Recently described interactions between the CTD and other cellular proteins, including YY-1, AP-2, BRCA-1, TFII-I, and Miz-1, suggest levels of regulatory complexity beyond Max in controlling DNA recognition by c-Myc. The N-terminal domain (NTD), which includes the evolutionarily conserved and functionally crucial Myc Box sequences (MB1 and MB2), contains the transcription activation domain (TAD) of c-Myc as well as regions required for transcriptional repression, cell cycle regulation, transformation, and apoptosis. In addition to interaction with the retinoblastoma family protein p107, the NTD has been shown to interact with alpha-tubulin and the novel adaptor proteins Binl, MM-1, Pam, TRRAP, and AMY-1. The structure of these proteins and their effects on c-Myc actions suggest links to the transcriptional regulatory machinery as well as to cell cycle regulation, chromatin modeling, and apoptosis. Investigations of this emerging NTD-based network may reveal how c-Myc is regulated and how it affects cell fate, as well as providing tools to distinguish the physiological roles of various Myc target genes. (+info)
The basic region/helix-loop-helix/leucine zipper domain of Myc proto-oncoproteins: function and regulation.
(19/1416)
A large body of evidence has been accumulated that demonstrates dominant effects of Myc proto-oncoproteins on different aspects of cellular growth. Myc is one of the few proteins that is sufficient to drive resting cells into the cell cycle and promote DNA synthesis. In line with this finding is that the constitutive expression of Myc in cells blocks their differentiation. These growth stimulating properties are most likely responsible for Myc's ability to initiate and promote tumor formation. Interestingly Myc can also sensitize cells to apoptosis, suggesting that this protein is part of a life-and-death switch. Molecularly Myc functions as a transcriptional regulator that needs to heterodimerize with Max to exert the biological activities described above and to regulate gene transcription. Myc and Max are just two members of a growing family of proteins referred to as the Myc/Max/Mad network. A hallmark of these proteins is that they possess a C-terminal basic region/helix-loop-helix/leucine zipper domain (bHLHZip). The bHLHZip domain specifies dimerization within the network and determines sequence specific DNA binding. Importantly this domain together with the N-terminal transactivation domain is essential for Myc biology. Here we have summarized the structural, functional, and regulatory aspects of the bHLHZip domain of Myc proteins. (+info)
Two MAD tails: what the recent knockouts of Mad1 and Mxi1 tell us about the MYC/MAX/MAD network.
(20/1416)
Members of the MAD/MXI protein family heterodimerize with MAX and repress transcription by recruiting a chromatin-modifying co-repressor complex to specific DNA target genes. Repression mediated by MAD is thought to antagonize the transcriptional activation and proliferation-promoting functions of MYC-MAX heterodimers. Because they are induced during differentiation, it has been suggested that MAD proteins act to limit cell proliferation during terminal differentiation. There is also controversial evidence that these proteins may function as tumor suppressors. Recently, targeted gene deletions of two members of this gene family, Mad1 and Mxi1, have been carried out in mice. Although these animals display what appear to be quite different phenotypes, further analysis supports the view that both these proteins function in cell-cycle exit during terminal differentiation, and that at least MXI1 can act as a tumor suppressor. (+info)
Sin1: an evolutionarily conserved component of the eukaryotic SAPK pathway.
(21/1416)
The fission yeast Sty1/Spc1 mitogen-activated protein (MAP) kinase is a member of the eukaryotic stress-activated MAP kinase (SAPK) family. We have identified a protein, Sin1, that interacts with Sty1/Spc1 which is a member of a new evolutionarily conserved gene family. Cells lacking Sin1 display many, but not all, of the phenotypes of cells lacking the Sty1/Spc1 MAP kinase including sterility, multiple stress sensitivity and a cell-cycle delay. Sin1 is phosphorylated after stress but this is not Sty1/Spc1-dependent. Importantly, Sin1 is not required for activation of Sty1/Spc1 but is required for stress-dependent transcription via its substrate, Atf1. We find that in the absence of Sin1, Sty1/Spc1 appears to translocate to the nucleus but Atf1 is not fully phosphorylated and becomes unstable in response to environmental stress. Sin1 is also required for effective transcription via the AP-1 factor Pap1 but does not prevent its nuclear translocation. Remarkably chimaeric fusions of sin1 with chicken sin1 sequences rescue loss of sin1 function. We conclude that Sin1 is a novel component of the eukaryotic SAPK pathway. (+info)
Plo1 kinase recruitment to the spindle pole body and its role in cell division in Schizosaccharomyces pombe.
(22/1416)
Polo kinases execute multiple roles during cell division. The fission yeast polo related kinase Plo1 is required to assemble the mitotic spindle, the prophase actin ring that predicts the site for cytokinesis and for septation after the completion of mitosis (Ohkura et al., 1995; Bahler et al., 1998). We show that Plo1 associates with the mitotic but not interphase spindle pole body (SPB). SPB association of Plo1 is the earliest fission yeast mitotic event recorded to date. SPB association is strong from mitotic commitment to early anaphase B, after which the Plo1 signal becomes very weak and finally disappears upon spindle breakdown. SPB association of Plo1 requires mitosis-promoting factor (MPF) activity, whereas its disassociation requires the activity of the anaphase-promoting complex. The stf1.1 mutation bypasses the usual requirement for the MPF activator Cdc25 (Hudson et al., 1990). Significantly, Plo1 associates inappropriately with the interphase SPB of stf1.1 cells. These data are consistent with the emerging theme from many systems that polo kinases participate in the regulation of MPF to determine the timing of commitment to mitosis and may indicate that pole association is a key aspect of Plo1 function. Plo1 does not associate with the SPB when septation is inappropriately driven by deregulation of the Spg1 pathway and remains SPB associated if septation occurs in the presence of a spindle. Thus, neither Plo1 recruitment to nor its departure from the SPB are required for septation; however, overexpression of plo1+ activates the Spg1 pathway and causes transient Cdc7 recruitment to the SPB and multiple rounds of septation. (+info)
Specificity of DNA binding of the c-Myc/Max and ARNT/ARNT dimers at the CACGTG recognition site.
(23/1416)
Basic helix-loop-helix proteins that interact with the DNA recognition site CACGTG include the c-Myc/Max heterodimer and the ARNT (Ahreceptornucleartranslocator) homodimer. We have utilized a PCR-based protocol to identify high affinity binding sites of either the c-Myc/Max or ARNT/ARNT dimers and analyzed the ability of these dimers to interact with their derived consensus sequences and activate genes. chi(2)analysis of the selected DNA recognition sites revealed that DNA binding of the ARNT homodimer is symmetric, resulting in the consensus sequence RTCACGTGAY. Gel shift analysis demonstrated that the flanking nucleotides play an important role in dictating DNA binding affinity of the ARNT homodimer. These flanking sequences also regulate the ability of ARNT to competitively displace the c-Myc/Max heterodimer from a CACGTG-containing sequence. However, transient transfection analyses in CV-1 cells revealed that ARNT and c-Myc/Max exhibited similar abilities to activate transcription through each other's consensus sequences. Taken together, these results indicate that although binding affinity of these dimers for the CACGTG core sequences may be differentially influenced by flanking nucleotides, transcriptional activity may also be determined by other factors, such as cellular concentrations of these proteins and their co-activators. (+info)
Identification of cis-acting elements important for expression of the starch-branching enzyme I gene in maize endosperm.
(24/1416)
The genes encoding the starch-branching enzymes (SBE) SBEI, SBEIIa, and SBEIIb in maize (Zea mays) are differentially regulated in tissue specificity and during kernel development. To gain insight into the regulatory mechanisms controlling their expression, we analyzed the 5'-flanking sequences of Sbe1 using a transient gene expression system. Although the 2.2-kb 5'-flanking sequence between -2,190 and +27 relative to the transcription initiation site was sufficient to promote transcription, the addition of the transcribed region between +28 and +228 containing the first exon and intron resulted in high-level expression in suspension-cultured maize endosperm cells. A series of 5' deletion and linker-substitution mutants identified two critical positive cis elements, -314 to -295 and -284 to -255. An electrophoretic mobility-shift assay showed that nuclear proteins prepared from maize kernels interact with the 60-bp fragment containing these two elements. Expression of the Sbe1 gene is regulated by sugar concentration in suspension-cultured maize endosperm cells, and the region -314 to -145 is essential for this effect. Interestingly, the expression of mEmBP-1, a bZIP transcription activator, in suspension-cultured maize endosperm cells resulted in a 5-fold decrease in Sbe1 promoter activity, suggesting a possible regulatory role of the G-box present in the Sbe1 promoter from -227 to -220. (+info)