bluuebunny/crossref_metadata_2025 huggingface dataset:
AP-1, ETS, and transcriptional silencers regulate retinoic acid-dependent induction of keratin 18 in embryonic cells.
Authors: Pankov R , Neznanov N , Umezawa A , Oshima R G
Published: October 2015

The differentiation of both embryonal carcinoma (EC) and embryonic stem (ES) cells can be triggered in culture by exposure to retinoic acid and results in the transcriptional induction of both the endogenous mouse keratin 18 (mK18) intermediate filament gene and an experimentally introduced human keratin 18 (K18) gene as well as a variety of other markers characteristic of extraembryonic endoderm. The induction of K18 in EC cells is limited, in part, by low levels of ETS and AP-1 transcription factor activities which bind to sites within a complex enhancer element located within the first intron of K18. RNA levels of ETS-2, c-Jun, and JunB increase upon the differentiation of ES cells and correlate with increased expression of K18. Occupancy of the ETS site, detected by in vivo footprinting methods, correlates with K18 induction in ES cells. In somatic cells, the ETS and AP-1 elements mediate induction by a variety of oncogenes associated with the ras signal transduction pathway. In EC cells, in addition to the induction by these limiting transcription factors, relief from negative regulation is mediated by three silencer elements located within the first intron of the K18 gene. These silencer elements function in F9 EC cells but not their differentiated derivatives, and their activity is correlated with proteins in F9 EC nuclei which bind to the silencers and are reduced in the nuclei of differentiated F9 cells. The induction of K18, associated with the differentiation of EC cells to extraembryonic endoderm, is due to a combination of relief from negative regulation and activation by members of the ETS and AP-1 transcription factor families.
Transactivation of the human p53 tumor suppressor gene by c-Myc/Max contributes to elevated mutant p53 expression in some tumors.
Authors: Roy B , Beamon J , Balint E , Reisman D
Published: October 2015

Elevated levels of mutant forms of the p53 tumor suppressor are a hallmark of many transformed cells. Multiple mechanisms such as increased stability of the protein and increased transcription of the gene can account for elevated p53 expression. Recent findings indicate that c-Myc/Max heterodimers can bind to an essential CA(C/T)GTG-containing site in the p53 promoter and elevate its expression. We have addressed the possibility that elevated mutant p53 expression is due to deregulated c-Myc expression. Here we demonstrate that the human p53 promoter is transactivated by high c-Myc expression and repressed by high Max expression. In examining the relative levels of c-Myc and p53 in human Burkitt's lymphomas and other B-lymphoid lines, we found that there is a correlation between the levels of c-Myc protein and p53 mRNA expression. In particular, cells that express very low levels of c-Myc protein also express low levels of p53 mRNA, while cells that express high levels of c-Myc tend to express high levels of p53 mRNA. To determine whether the p53 gene can be a target for c-Myc in vivo, we assayed the effects of antisense c-myc RNA on the levels of endogenous p53 mRNA. The results indicate that the presence of antisense c-myc RNA leads to a reduction in the levels of c-Myc protein, p53 mRNA, and expression from the p53 promoter. Taken together, our findings support a direct role for c-Myc in elevating expression of the mutant p53 gene in some tumors.
Analysis of the DNA-binding and dimerization activities of Neurospora crassa transcription factor NUC-1.
Authors: Peleg Y , Metzenberg R L
Published: October 2015

NUC-1, a positive regulatory protein of Neurospora crassa, controls the expression of several unlinked target genes involved in phosphorus acquisition. The carboxy-terminal end of the NUC-1 protein has sequence similarity to the helix-loop-helix family of transcription factors. Bacterially expressed and in vitro-synthesized proteins, which consist of the carboxy-terminal portion of NUC-1, bind specifically to upstream sequences of two of its target genes, pho2+ and pho-4+. These upstream sequences contain the core sequence, CACGTG, a target for many helix-loop-helix proteins. A large loop region (47 amino acids) separates the helix I and helix II domains. Mutations and deletion within the loop region did not interfere with the in vitro or in vivo functions of the protein. Immediately carboxy-proximal to the helix II domain, the NUC-1 protein contains an atypical zipper domain which is essential for function. This domain consists of a heptad repeat of alanine and methionine rather than leucine residues. Analysis of mutant NUC-1 proteins suggests that the helix II and the zipper domains are essential for the protein dimerization, whereas the basic and the helix I domains are involved in DNA binding. The helix I domain, even though likely to participate in dimer formation while NUC-1 is bound to DNA, is not essential for in vitro dimerization.
Stress resistance in Saccharomyces cerevisiae is strongly correlated with assembly of a novel type of multiubiquitin chain.
Authors: Arnason T , Ellison M J
Published: October 2015

The covalent attachment of ubiquitin (Ub) to short-lived or damaged proteins is believed to be the signal that initiates their selective degradation. In several cases, it has been shown that the proteolytic signal takes the form of a multi-Ub chain in which successive Ub molecules are linked tandemly at lysine 48 (K-48). Here we show that Ub molecules can be linked together in vivo at two other lysine positions, lysine 29 (K-29) and lysine 63 (K-63). The formation of these alternative linkages is strongly dependent on the presence of the stress-related Ub conjugating enzymes UBC4 and UBC5. Furthermore, expression of Ub carrying a K-63 to arginine 63 substitution in a strain of Saccharomyces cerevisiae that is missing the poly-Ub gene, UBI4, fails to compensate for the stress defects associated with these cells. Taken together, these results suggest that the formation of multi-Ub chains involving K-63 linkages plays an important role in the yeast stress response. In broader terms, these results also suggest that Ub is a versatile signal in which different Ub chain configurations are used for different functions.
A family of complex tandem DNA repeats in the telomeres of Chironomus pallidivittatus.
Authors: Zhang Y J , Kamnert I , López C C , Cohn M , Edström J E
Published: October 2015

A family of 340-bp tandem telomere-associated DNA repeats is present in 50- to 200-kb blocks in seven of the eight paired chromosome ends in Chironomus pallidivittatus. It consists of four main subfamilies, differing from each other by small clusters of mutations. This differentiation may reflect different functional roles for the repeats. Here we find that one subfamily, D3, is consistently localized most peripherally and extends close to the ends of the chromosomes, as shown by its sensitivity to the exonuclease Bal 31. The amounts of D3 are highly variable between individuals. The repeat characteristic for D3 forms a segment with pronounced dyad symmetry, which in single-strand form would give rise to a hairpin. Evidence from an interspecies comparison suggests that a similar structure is the result of selective forces. Another subfamily, M1, is present more proximally in a subgroup of telomeres characterized by a special kind of repeat variability. Thus, a complex block with three kinds of subfamilies may occupy different M1 telomeres depending on the stock of animals. We conclude that subfamilies are differentially distributed between and within telomeres and are likely to serve different functions.
Multiple positive and negative regulatory elements in the promoter of the mouse homeobox gene Hoxb-4.
Authors: Gutman A , Gilthorpe J , Rigby P W
Published: October 2015

Mouse Hoxb-4 (Hox-2.6) is a homeobox gene that belongs to a family which also includes Hoxa-4, Hoxc-4, and Hoxd-4 and that is related to the Deformed gene in Drosophila melanogaster. We have determined the sequence of 1.2 kb of 5' flanking DNA of mouse Hoxb-4 and by nuclease S1 and primer extension experiments identified two transcription start sites, P1 and P2, 285 and 207 nucleotides upstream of the ATG initiator codon, respectively. We have shown that this region harbors two independent promoters which drive CAT expression in several different cell lines with various efficiencies, suggesting that they are subject to cell-type-specific regulation. Through detailed mutational analysis, we have identified several cis-regulatory elements, located upstream and downstream of the transcription start sites. They include two cell-type-specific negative regulatory elements, which are more active in F9 embryonal carcinoma cells than in neuroblastoma cells (regions a and d at -226 to -186 and +169 to +205, respectively). An additional negative regulatory element has been delimited (region b between +22 and +113). Positive regulation is achieved by binding of HoxTF, a previously unknown factor, to the sequence GCCATTGG (+148 to +155) that is essential for efficient Hoxb-4 expression. We have also defined the minimal promoter sequences and found that they include two 12-bp initiator elements centered around each transcription start site. The complex architecture of the Hoxb-4 promoter provides the framework for fine-tuned transcriptional regulation during embryonic development.
Heat shock transcription factor activates yeast metallothionein gene expression in response to heat and glucose starvation via distinct signalling pathways.
Authors: Tamai K T , Liu X , Silar P , Sosinowski T , Thiele D J
Published: October 2015

Metallothioneins constitute a class of low-molecular-weight, cysteine-rich metal-binding stress proteins which are biosynthetically regulated at the level of gene transcription in response to metals, hormones, cytokines, and other physiological and environmental stresses. In this report, we demonstrate that the Saccharomyces cerevisiae metallothionein gene, designated CUP1, is transcriptionally activated in response to heat shock and glucose starvation through the action of heat shock transcription factor (HSF) and a heat shock element located within the CUP1 promoter upstream regulatory region. CUP1 gene activation in response to both stresses occurs rapidly; however, heat shock activates CUP1 gene expression transiently, whereas glucose starvation activates CUP1 gene expression in a sustained manner for at least 2.5 h. Although a carboxyl-terminal HSF transcriptional activation domain is critical for the activation of CUP1 transcription in response to both heat shock stress and glucose starvation, this region is dispensable for transient heat shock activation of at least two genes encoding members of the S. cerevisiae hsp70 family. Furthermore, inactivation of the chromosomal SNF1 gene, encoding a serine-threonine protein kinase, or the SNF4 gene, encoding a SNF1 cofactor, abolishes CUP1 transcriptional activation in response to glucose starvation without altering heat shock-induced transcription. These studies demonstrate that the S. cerevisiae HSF responds to multiple, distinct stimuli to activate yeast metallothionein gene transcription and that these stimuli elicit responses through nonidentical, genetically separable signalling pathways.
RNA editing and mitochondrial genomic organization in the cryptobiid kinetoplastid protozoan Trypanoplasma borreli.
Authors: Maslov D A , Simpson L
Published: October 2015

The bodonids and cryptobiids represent an early diverged sister group to the trypanosomatids among the kinetoplastid protozoa. The trypanosome type of uridine insertion-deletion RNA editing was found to occur in the cryptobiid fish parasite Trypanoplasma borreli. A pan-edited ribosomal protein, S12, and a novel 3'- and 5'-edited cytochrome b, in addition to an unedited cytochrome oxidase III gene and an apparently unedited 12S rRNA gene, were found in a 6-kb fragment of the 80- to 90-kb mitochondrial genome. The gene order differs from that in trypanosomatids, as does the organization of putative guide RNA genes; guide RNA-like molecules are transcribed from tandemly repeated 1-kb sequences organized in 200- and 170-kb molecules instead of minicircles. The presence of pan-editing in this lineage is consistent with an ancient evolutionary origin of this process.
Transposition of the yeast retroviruslike element Ty3 is dependent on the cell cycle.
Authors: Menees T M , Sandmeyer S B
Published: October 2015

Host cell cycle genes provide important functions to retroviruses and retroviruslike elements. To define some of these functions, the cell cycle dependence of transposition of the yeast retroviruslike element Ty3 was examined. Ty3 is unique among retroviruslike elements because of the specificity of its integration, which occurs upstream of genes transcribed by RNA polymerase III. A physical assay for Ty3 transposition which takes advantage of this position-specific integration was developed. The assay uses PCR to amplify a product of Ty3 integration into a target plasmid that carries a modified tRNA gene. By using the GAL1 upstream activating sequence to regulate expression of Ty3, transposition was detected within one generation of cell growth after Ty3 transcription was initiated. This physical assay was used to show that Ty3 did not transpose when yeast cells were arrested in G1 during treatment with the mating pheromone alpha-factor. The restriction of transposition was not due to changes in transcription of either Ty3 or tRNA genes or to aspects of the mating pheromone response unrelated to cell cycle arrest. The block of the Ty3 life cycle was reversed when cells were released from G1 arrest. Examination of Ty3 intermediates during G1 arrest indicated that Ty3 viruslike particles were present but that reverse transcription of the Ty3 genomic RNA into double-stranded DNA had not occurred. In G1, the Ty3 life cycle is blocked after particle assembly but before the completion of reverse transcription.
Positive and negative regulation of cell proliferation by E2F-1: influence of protein level and human papillomavirus oncoproteins.
Authors: Melillo R M , Helin K , Lowy D R , Schiller J T
Published: October 2015

E2F-1 is a member of a family of transcription factors implicated in the activation of genes required for the progression through the S phase of the cell cycle. We have examined the biological activities of E2F-1 with short-term colony-forming assays and long-term immortalization assays. High levels of E2F-1, produced by transfection of the E2F-1 cDNA under the control of a strong promoter, reduced colony formation in normal human foreskin keratinocytes (NHFKs). This inhibition could not be overcome by wild-type human papillomavirus type 16 (HPV16) E6 and E7, two proteins which cooperate to immortalize NHFKs, or by a transdominant p53 mutant. High levels of E2F-1 also inhibited growth of primary and established fibroblasts. The growth-inhibitory activity required the DNA binding function of E2F-1 but not its transactivation or pRB binding activities. A positive role for lower levels of E2F-1 in NHFK immortalization was established by examining the ability of E2F-1 to complement HPV16 E7 mutants that were unable to cooperate with HPV16 E6 to immortalize NHFKs. Although E2F-1 was unable by itself to cooperate with E6, it did, in conjunction with E6, complement a p24GLY mutant of E7 that is defective for immortalization and binding of pRB and pRB-related proteins. By contrast, E2F-1 was unable to complement two other E7 mutants, p2PRO and p31/32ARG/PRO, which are also defective in the immortalization assay, although their proteins display wild-type binding of pRB in vitro. Since the binding of E7 to pRB results in disruption of pRB-E2F interaction and release of transcriptionally active E2F, the data support the hypothesis that binding of pRB by E7 and the consequence increase in E2F, the data support the hypothesis that binding of pRB by E7 and the consequence increase in E3F activity are important but not sufficient for E7-induced keratinocyte immortalization.
Targeted expression of the E6 and E7 oncogenes of human papillomavirus type 16 in the epidermis of transgenic mice elicits generalized epidermal hyperplasia involving autocrine factors.
Authors: Auewarakul P , Gissmann L , Cid-Arregui A
Published: October 2015

The E6 and E7 early genes of human papillomavirus type 16 have been shown in vitro to play a central role in the transforming capability of this virus. To explore their effects on differentiating epithelial cells in vivo, we used a bovine cytokeratin 10 (K10) promoter to target the expression of E6 and E7 to the suprabasal layers of the epidermis of transgenic mice. In two different lines of mice efficiently expressing the transgene, animals displayed generalized epidermal hyperplasia, hyperkeratosis and parakeratosis in the skin and the forestomach, both known to be sites of K10 expression. Northern (RNA) blot analysis revealed high levels of E6 and E7 transcripts, and in situ hybridizations localized these transcripts to the suprabasal strata of epidermis. In vivo labeling of proliferating cells showed two distinct effects of E6 and E7 expression in the epidermis: (i) an increase in the number of growing cells in the undifferentiated basal layer and (ii) abnormal proliferation of differentiated cells in the suprabasal strata. The expression of c-myc in the skin of transgenics was higher than that in control animals. The induction of c-myc transcription by topical application of tetradecanoyl phorbol acetate was prevented by simultaneous treatment with transforming growth factor beta 1 in nontransgenic skin but not in transgenic skin. In addition, transforming growth factor alpha was found to be overexpressed in the suprabasal layers of the transgenic epidermis. These findings suggest that autocrine mechanisms are involved in the development and maintenance of epidermal hyperplasia. Animals of both lines developed papillomas in skin sites exposed to mechanical irritation and wounding, suggesting that secondary events are necessary for progression to neoplasia. Collectively, these results provide new insights into the tumor promoter activities of human papillomavirus type 16 in epithelial cells in vivo.
Type I adenylyl cyclase functions as a coincidence detector for control of cyclic AMP response element-mediated transcription: synergistic regulation of transcription by Ca2+ and isoproterenol.
Authors: Impey S , Wayman G , Wu Z , Storm D R
Published: October 2015

Studies carried out with mammals and invertebrates suggest that Ca(2+)-sensitive adenylyl cyclases may be important for neuroplasticity. Long-term potentiation in the hippocampus requires increases in intracellular Ca2+ which are accompanied by elevated cyclic AMP (cAMP). Furthermore, activation of cAMP-dependent protein kinase is required for the late stage of long-term potentiation in the CA1 region of the hippocampus, which is also sensitive to inhibitors of transcription. Therefore, some forms of synaptic plasticity may require coordinate regulation of transcription by Ca2+ and cAMP. In this study, we demonstrate that the expression of type I adenylyl cyclase in HEK-293 cells allows Ca2+ to stimulate reporter gene activity mediated through the cAMP response element. Furthermore, simultaneous activation by Ca2+ and isoproterenol caused synergistic stimulation of transcription in HEK-293 cells and cultured neurons. We propose that Ca2+ and neurotransmitter stimulation of type I adenylyl cyclase may play a role in synaptic plasticity by generating optimal cAMP signals for regulation of transcription.
The Ikaros gene encodes a family of functionally diverse zinc finger DNA-binding proteins.
Authors: Molnár A , Georgopoulos K
Published: October 2015

We previously described the lymphocyte-restricted Ikaros gene encoding a zinc finger DNA-binding protein as a potential regulator of lymphocyte commitment and differentiation. Here, we report the isolation of four additional Ikaros transcripts, products of alternate splicing that encode functionally diverse proteins. The Ikaros proteins contain unique combinations of zinc finger modules that dictate their overall sequence specificity and affinity. The Ik-1 and Ik-2 proteins can both bind, albeit with different affinities, to the same recognition sequences present in a number of lymphocyte-specific regulatory elements. The Ik-3 and the Ik-4 proteins interact only with a subset of these motifs. The Ik-1 and Ik-2 proteins can strongly stimulate transcription, whereas Ik-3 and Ik-4 are weak activators. Significantly, the transcription activation potential of the Ikaros proteins correlates with their subcellular localization. Upon ectopic expression of the Ikaros isoforms in nonlymphoid cells, Ik-1 and Ik-2 localize to the nucleus, whereas Ik-3 and Ik-4 are predominantly found in the cytoplasm. The Ikaros isoforms are expressed differentially in lymphocytes: Ik-1 and Ik-2 mRNAs are the predominating forms, and Ik-4 is present in significant amounts only in early T-cell progenitors, whereas Ik-3 and Ik-5 transcripts are expressed at relatively low levels throughout lymphocyte development. The ability of the Ikaros gene to generate functionally diverse proteins that may participate in distinct regulatory pathways substantiates its role as a master regulator during lymphocyte development.
Transformation properties of the E2a-Pbx1 chimeric oncoprotein: fusion with E2a is essential, but the Pbx1 homeodomain is dispensable.
Authors: Monica K , LeBrun D P , Dedera D A , Brown R , Cleary M L
Published: October 2015

The t(1;19) chromosomal translocation in acute lymphoblastic leukemias creates chimeric E2a-Pbx1 oncoproteins that can act as DNA-binding activators of transcription. A structural analysis of the functional domains of E2a-Pbx1 showed that portions of both E2a and Pbx1 were essential for transformation of NIH 3T3 cells and transcriptional activation of synthetic reporter genes containing PBX1 consensus binding sites. Hyperexpression of wild-type or experimentally truncated Pbx1 proteins was insufficient for transformation, consistent with their inability to activate transcription. When fused with E2a, the Pbx-related proteins Pbx2 and Pbx3 were also transformation competent, demonstrating that all known members of this highly similar subfamily of homeodomain proteins have latent oncogenic potential. The oncogenic contributions of E2a to the chimeras were localized to transactivation motifs AD1 and AD2, as their mutation significantly impaired transformation. Either the homeodomain or Pbx1 amino acids flanking this region could mediate transformation when fused to E2a. However, the homeodomain was not essential for transformation, since a mutant E2a-Pbx1 protein (E2a-Pbx delta HD) lacking the homeodomain efficiently transformed fibroblasts and induced malignant lymphomas in transgenic mice. Thus, transformation mediated by the chimeric oncoprotein E2a-Pbx1 is absolutely dependent on motifs acquired from E2a but the Pbx1 homeodomain is optional. The latter finding suggests that E2a-Pbx1 may interact with cellular proteins that assist or mediate alterations in gene expression responsible for oncogenesis even in the absence of homeodomain-DNA interactions.
Use of double-replacement gene targeting to replace the murine alpha-lactalbumin gene with its human counterpart in embryonic stem cells and mice.
Authors: Stacey A , Schnieke A , McWhir J , Cooper J , Colman A , Melton D W
Published: October 2015

The mouse alpha-lactalbumin gene has been replaced with the human gene by two consecutive rounds of gene targeting in hypoxanthine phosphoribosyltransferase (HPRT)-deficient feeder-independent murine embryonic stem (ES) cells. One mouse alpha-lactalbumin allele was first replaced by an HPRT minigene which was in turn replaced by human alpha-lactalbumin. The end result is a clean exchange of defined DNA fragments with no other DNA remaining at the target locus. Targeted ES cells at each stage remained capable of contributing efficiently to the germ line of chimeric animals. Double replacement using HPRT-deficient ES cells and the HPRT selection system is therefore a powerful and flexible method of targeting specific alterations to animal genes. A typical strategy for future use would be to generate a null mutation which could then be used to produce multiple second-step alterations at the same locus.
Anisomycin and rapamycin define an area upstream of p70/85S6k containing a bifurcation to histone H3-HMG-like protein phosphorylation and c-fos-c-jun induction.
Authors: Kardalinou E , Zhelev N , Hazzalin C A , Mahadevan L C
Published: October 2015

Anisomycin, a translational inhibitor, synergizes with growth factors and phorbol esters to superinduce c-fos and c-jun by a number mechanisms, one of which is its ability to act as a potent signalling agonist, producing strong, prolonged activation of the same nuclear responses as epidermal growth factor or tetradecanoyl phorbol acetate. These responses include the phosphorylation of pp33, which exists in complexed and chromatin-associated forms, and of histone H3 and an HMG-like protein. By peptide mapping and microsequencing, we show here that pp33 is the phosphoprotein S6, present in ribosomes and in preribosomes in the nucleolus. Ablation of epidermal growth factor-, tetradecanoyl phorbol acetate-, or anisomycin-stimulated S6 phosphorylation by using the p70/85S6k inhibitor rapamycin has no effect on histone H3 and HMG-like protein phosphorylation or on the induction and superinduction of c-fos and c-jun. Further, [35S]methionine-labelling and immunoprecipitation studies show that the ablation of S6 phosphorylation has no discernible effect on translation in general or translation of newly induced c-fos transcripts. Finally, we show that anisomycin augments and prolongs S6 phosphorylation not by blocking S6 phosphatases but by sustained activation of p70/85S6k. These results suggest the possible use of anisomycin and rapamycin to define upstream and downstream boundaries of an area of signalling above p70/85S6k which contains a bifurcation that produces histone H3-HMG-like protein phosphorylation and c-fos-c-jun induction in the nucleus.
Activation of phospholipase C-gamma 1 through transfected platelet-derived growth factor receptor enhances interleukin 2 production upon antigen stimulation in a T-cell line.
Authors: Nakano H , Ohno H , Saito T
Published: October 2015

Phospholipase C-gamma 1 (PLC gamma 1) plays an important role in the signal transduction pathway by producing second messengers. However, the activation mechanism of PLC gamma 1 and the role of the phosphatidylinositol pathway for interleukin 2 (IL-2) production in T lymphocytes remain to be determined. To analyze the functional role of this pathway in T cells, we expressed an epidermal growth factor receptor (EGF) or platelet-derived growth factor (PDGF) receptor (EGF-R or PDGF-R), both of which are known to directly activate PLC gamma 1 in fibroblasts, into a murine T-cell hybridoma. Both receptors were expressed on the cell surface and caused tyrosine phosphorylation of multiple substrates, including the receptor itself, upon ligand binding. While EGF stimulation did not either cause phosphorylation of PLC gamma 1 or induce Ca2+ mobilization in the EGF-R transfectant in this system, PDGF treatment induced tyrosine phosphorylation of PLC gamma 1 and Ca2+ mobilization in the PDGF-R transfectant. Stimulation through PDGF-R enhanced IL-2 production upon antigen stimulation of the transfectants, although PDGF treatment alone did not induce IL-2 production. These results suggest that activation of the phosphatidylinositol pathway affects the downstream pathway to IL-2 production but is not sufficient to produce IL-2 and that cooperation with signals from tyrosine kinase cascades is required for IL-2 production.
HEN1 encodes a 20-kilodalton phosphoprotein that binds an extended E-box motif as a homodimer.
Authors: Brown L , Baer R
Published: October 2015

HEN1 and HEN2 encode neuron-specific polypeptides that contain the basic helix-loop-helix (bHLH) motif, a protein dimerization and DNA-binding domain common to several known transcription factors. We now describe characteristics of the HEN1 gene product that are consistent with its postulated role as a transcription factor that functions during development of the mammalian nervous system. Thus, transcription of the HEN1 gene is activated upon the induction of neural differentiation in PC12 cells by nerve growth factor. HEN1 encodes a 20-kDa polypeptide (pp20HEN1) that is phosphorylated exclusively at serine residues and forms dimeric bHLH complexes either by self-association or by heterologous interaction with the E2A gene products (E12 or E47). The resultant HEN1/HEN1 homodimers and HEN1/E2A heterodimers bind DNA in a sequence-specific manner. Moreover, a binding site selection procedure revealed that HEN1-HEN1 homodimers preferentially recognize E-box motifs represented by an 18-bp consensus sequence (GGGNCG CAGCTGCGNCCC). The E-box half-site recognized by HEN1 polypeptides (GGGNCGCAG) is distinct from those of other known bHLH proteins, suggesting that HEN1 binds, an regulates the transcription of, a unique subset of target genes during neural development.
Human T-cell leukemia virus type I Tax associates with and is negatively regulated by the NF-kappa B2 p100 gene product: implications for viral latency.
Authors: Béraud C , Sun S C , Ganchi P , Ballard D W , Greene W C
Published: October 2015

Human T-cell leukemia virus type I (HTLV-I) is the etiologic agent of the adult T-cell leukemia, an aggressive and often fatal malignancy of activated human CD4 T cells. HTLV-I encodes an essential 40-kDa protein termed Tax that not only transactivates the long terminal repeat of this retrovirus but also induces an array of cellular genes. Tax-mediated transformation of T cells likely involves the deregulated expression of various cellular genes that normally regulate lymphocyte growth produced by altered activity of various endogenous host transcription factors. In particular, Tax is capable of modulating the expression or activity of various host transcription factors, including members of the NF-kappa B/Rel and CREB/ATF families, as well as the cellular factors HEB-1 and p67SRF. An additional distinguishing characteristic of HTLV-I infection is the profound state of viral latency that is present in circulating primary leukemic T cells. In this study, we demonstrate that HTLV-I Tax can physically associate with p100, the product of the Rel-related NF-kappa B2 gene, both in transfected cells and in HTLV-I-infected leukemic T-cell lines. Furthermore, the physical interaction of Tax with p100 leads to the inhibition of Tax-induced activation of the HTLV-I and human immunodeficiency virus type 1 long terminal repeats, reflecting p100-mediated cytoplasmic sequestration of the normally nuclearly expressed Tax protein. In contrast, a mutant of Tax that selectively fails to activate nuclear NF-kappa B expression does not associate with p100. Together, these results suggest that the cytoplasmic interplay of Tax and p100 may play an important role in the initiation and maintenance of HTLV-1 latency observed in adult T-cell leukemia.
Nucleotide sequence of the yeast STE14 gene, which encodes farnesylcysteine carboxyl methyltransferase, and demonstration of its essential role in a-factor export.
Authors: Sapperstein S , Berkower C , Michaelis S
Published: October 2015

Eukaryotic proteins initially synthesized with a C-terminal CAAX motif (C is Cys, A is aliphatic, and X can be one of several amino acids) undergo a series of modifications involving isoprenylation of the Cys residue, proteolysis of AAX, and alpha-carboxyl methyl esterification of the newly formed isoprenyl cysteine. We have previously demonstrated that STE14 encodes the enzyme which mediates carboxyl methylation of the Saccharomyces cerevisiae CAAX proteins a-factor, RAS1, and RAS2. Here we report the nucleotide sequence of STE14, which indicates that STE14 encodes a protein of 239 amino acids, predicted to contain multiple membrane-spanning segments. Mapping data indicate that STE14 resides on chromosome IV, tightly linked to ADE8. By analysis of ste14 null alleles, we demonstrated that MATa ste14 mutants are unable to mate but are viable and exhibit no apparent growth defects. Additional analysis of ste14 ras 1 and ste14 ras2 double mutants, which grow normally, reinforces our previous conclusion that RAS function is not significantly influenced by its methylation status. We examine a-factor biogenesis in a ste14 null mutant by metabolic labeling and immunoprecipitation and demonstrate that although proteolytic processing and membrane localization of a-factor are normal, the ste14 null mutant exhibits a profound block in a-factor export. This observation suggests that the methyl group is likely to be a critical recognition determinant for the a-factor transporter, STE6, thus providing insight into the substrate specificity of STE6 and also supporting the hypothesis that carboxyl methylation can have a dramatic impact on protein-protein interactions.
The carboxy-terminal region of mammalian HSP90 is required for its dimerization and function in vivo.
Authors: Minami Y , Kimura Y , Kawasaki H , Suzuki K , Yahara I
Published: October 2015

The majority of mouse HSP90 exists as alpha-alpha and beta-beta homodimers. Truncation of the 15-kDa carboxy-terminal region of mouse HSP90 by digestion with the Ca(2+)-dependent protease m-calpain caused dissociation of the dimer. When expressed in a reticulocyte lysate, the full-length human HSP90 alpha formed a dimeric form. A plasmid harboring human HSP90 alpha cDNA was constructed so that the carboxy-terminal 49 amino acid residues were removed when translated in vitro. This carboxy-terminally truncated human HSP90 alpha was found to exist as a monomer. In contrast, loss of the 118 amino acid residues from the amino terminus of human HSP90 alpha did not affect its in vitro dimerization. Introduction of an expression plasmid harboring the full-length human HSP90 alpha complements the lethality caused by the double mutations of two HSP90-related genes, hsp82 and hsc82, in a haploid strain of Saccharomyces cerevisiae. The carboxy-terminally truncated human HSP90 alpha neither formed dimers in yeast cells nor rescued the lethal double mutant.
The Drosophila l(2)35Ba/nocA gene encodes a putative Zn finger protein involved in the development of the embryonic brain and the adult ocellar structures.
Authors: Cheah P Y , Meng Y B , Yang X , Kimbrell D , Ashburner M , Chia W
Published: October 2015

The Drosophila l(2)35Ba/nocA gene is involved in the development of the adult ocelli and the embryonic head. Mutations in this gene lead to at least two distinct phenotypes. Several larva lethal l(2)35Ba alleles cause both hypertrophy and mislocation of the embryonic supraesophageal ganglion (brain) to the dorsal surface of the embryo. A second class of mutant alleles (nocA) is homozygous viable, but the surviving adults either lack or have greatly reduced ocelli and associated bristles. The l(2)35Ba/nocA gene encodes an approximately 3.0-kb transcript doublet; all l(2)35Ba alleles which have been physically mapped delete or disrupt the transcribed region, whereas all of the viable nocA alleles are caused by gross chromosomal aberrations with breakpoints near the 3'-flanking region of the gene. Several nocA breakpoint alleles downregulate the level of l(2)35Ba/nocA transcripts in adults, and their defective ocellar phenotype also fails to be complemented by the lethal alleles, implying that l(2)35Ba and nocA are different phenotypic manifestations of mutations in the same gene. In the l(2)35Ba mutant embryos, cells from the procephalic lobe which normally migrate over and overlie the supraesophageal ganglion during head involution can become incorporated into the supraesophageal ganglion; many of these misplaced cells, which normally form the frontal sac, also adopt a neuronal fate. Sequence analysis of two full-length l(2)35Ba/nocA cDNAs with distinct polyadenylation sites shows that they encode the same deduced protein of 537 amino acids with a serine- and threonine-rich N-terminal region, two putative zinc finger motifs near the carboxyl terminus, and several alanine-rich domains. Consistent with the observed embryonic phenotype, l(2)35Ba/nocA shows a complex embryonic expression pattern which includes the procephalic lobe.
Analysis of an origin of DNA amplification in Sciara coprophila by a novel three-dimensional gel method.
Authors: Liang C , Gerbi S A
Published: October 2015

The replication origin region for DNA amplification in Sciara coprophila DNA puff II/9A was analyzed with a novel three-dimensional (3D) gel method. Our 3D gel method involves running a neutral/neutral 2D gel and then cutting out vertical gel slices from the area containing replication intermediates, rotating these slices 90 degrees to form the third dimension, and running an alkaline gel for each of the slices. Therefore, replication intermediates are separated into forks and bubbles and then are resolved into parental and nascent strands. We used this technique to determine the size of forks and bubbles and to confirm the location of the major initiation region previously mapped by 2D gels to a 1-kb region. Furthermore, our 3D gel analyses suggest that only one initiation event in the origin region occurs on a single DNA molecule and that the fork arc in the composite fork-plus-bubble pattern in neutral/neutral 2D gels does not result from broken bubbles.
Glucose-induced transcription of the insulin gene is mediated by factors required for beta-cell-type-specific expression.
Authors: Sharma A , Stein R
Published: October 2015

The insulin gene is expressed exclusively in pancreatic islet beta cells. The principal regulator of insulin gene transcription in the islet is the concentration of circulating glucose. Previous studies have demonstrated that transcription is regulated by the binding of trans-acting factors to specific cis-acting sequences within the 5'-flanking region of the insulin gene. To identify the cis-acting control elements within the rat insulin II gene that are responsible for regulating glucose-stimulated expression in the beta cell, we analyzed the effect of glucose on the in vivo expression of a series of transfected 5'-flanking deletion mutant constructs. We demonstrate that glucose-induced transcription of the rat insulin II gene is mediated by sequences located between -126 and -91 bp relative to the transcription start site. This region contains two cis-acting elements that are essential for directing pancreatic beta-cell-type-specific expression of the rat insulin II gene, the insulin control element (ICE; -100 to -91 bp) and RIPE3b1 (-115 to -107 bp). The gel mobility shift assay was used to determine whether the formation of the ICE- and RIPE3b1-specific factor-DNA element complexes were affected in glucose-treated beta-cell extracts. We found that RIPE3b1 binding activity was selectively induced by about eightfold. In contrast, binding to other insulin cis-acting element sequences like the ICE and RIPE3a2 (-108 to -99 bp) were unaffected by these conditions. The RIPE3b1 binding complex was shown to be distinct from the glucose-inducible factor that binds to an element located between -227 to -206 bp of the human and rat insulin I genes (D. Melloul, Y. Ben-Neriah, and E. Cerasi, Proc. Natl. Acad. Sci. USA 90:3865-3869, 1993). We have also shown that mannose, a sugar that can be metabolized by the beta cell, mimics the effects of glucose in the in vivo transfection assays and the in vitro RIPE3b1 binding assays. These results suggested that the RIPE3b1 transcription factor is a primary regulator of glucose-mediated transcription of the insulin gene. However, we found that mutations in either the ICE or the RIPE3b1 element reduced glucose-responsive expression from transfected 5'-flanking rat insulin II gene constructs. We therefore conclude that glucose-regulated transcription of the insulin gene is mediated by cis-acting elements required for beta-cell-type-specific expression.
A unique palindromic element mediates gamma interferon induction of mig gene expression.
Authors: Wong P , Severns C W , Guyer N B , Wright T M
Published: October 2015

To define the molecular mechanisms involved in the action of gamma interferon (IFN-gamma), we have analyzed the transcriptional regulation of the mig (monokine induced by gamma interferon) gene, a member of the platelet factor 4-interleukin-8 cytokine family that is expressed in murine macrophages specifically in response to IFN-gamma. Analysis of mig/CAT chimeric constructs transiently transfected into the RAW 264.7 mouse monocytic cell line revealed a unique IFN-gamma-responsive element (gamma RE-1). The sequence of this cis regulatory element defined by deletion analysis contains an imperfect inverted repeat extending 27 bp. Examination of mig/CAT constructs with mutations in gamma RE-1 revealed that the palindromic positions in the element were essential for activity. Consistent with its function as an enhancer, a single copy of gamma RE-1 conferred IFN-gamma inducibility to a heterologous (herpes simplex virus thymidine kinase) promoter. Exonuclease III protection assays demonstrated symmetrical protection of a mig promoter fragment centered about the gamma RE-1 palindromic sequence. Using the gel electrophoretic mobility shift assay, we identified a factor (gamma RF-1) present in nuclear extracts prepared from IFN-gamma-stimulated RAW 264.7 cells which binds to gamma RE-1. The activation of gamma RF-1 occurred rapidly (within 1 min) in response to IFN-gamma and was independent of protein synthesis. Similar to the expression of mig mRNA, the formation of gamma RF-1 was selectively induced by IFN-gamma and not IFN-alpha. The regulation of gene expression through gamma RF-1 and gamma RE-1 may explain the preferential activation of a subset of interferon-inducible genes by IFN-gamma.
Two different sequence elements within exon 4 are necessary for calcitonin-specific splicing of the human calcitonin/calcitonin gene-related peptide I pre-mRNA.
Authors: van Oers C C , Adema G J , Zandberg H , Moen T C , Baas P D
Published: October 2015

The calcitonin (CT)/calcitonin gene-related peptide I (CGRP-I) gene (CALC-I gene) is subject to alternative tissue-specific processing of its primary transcript. CT mRNA is the predominant mRNA produced in thyroid C cells, whereas CT gene-related peptide I mRNA is the main product in neurons of the central and peripheral nervous systems. The CT-specific exon 4 is surrounded by weak processing sites. In this study we have investigated whether exon 4 sequences are involved in the tissue-specific selection of the exon 4 splice acceptor site. The results indicate that two separate elements, termed A and B, in the 5' part of exon 4 are required for production of CT-specific RNA. These sequences are located between nucleotides 67 and 88 (A) and nucleotides 117 and 146 (B) relative to the 5' end of exon 4. Variation of the distance between these sequence elements and the 3' splice site of exon 4 does not change the processing choice. These sequence elements are functionally equivalent. CT-specific splicing requires the presence of both sequence A and B or duplicates of either sequence element in exon 4. The effect of these sequences on the RNA processing choice is overruled by mutation of the CT-specific uridine branch acceptor nucleotide into a commonly preferred adenosine residue.
Role of the histone amino termini in facilitated binding of a transcription factor, GAL4-AH, to nucleosome cores.
Authors: Vettese-Dadey M , Walter P , Chen H , Juan L J , Workman J L
Published: October 2015

Facilitated, "cooperative" binding of GAL4-AH to nucleosomal DNA occurred in response to inhibition from the core histone amino termini. The binding of GAL4-AH (which contains the DNA-binding and dimerization domains of GAL4) to nucleosome cores containing multiple binding sites initiated at the end of a nucleosome core and proceeded in a cooperative manner until all sites were occupied. However, following tryptic removal of the core histone amino termini, GAL4-AH binding appeared to be noncooperative, similar to binding naked DNA. Binding of GAL4-AH to nucleosomes bearing a single GAL4 site at different positions indicated that inhibition of GAL4 binding was largely mediated by the histone amino termini and primarily occurred at sites well within the core and not near the end. When the histone amino termini were intact, binding of GAL4-AH to sites near the center of a nucleosome core was greatly enhanced by the presence of additional GAL4 dimers bound to more-accessible positions. These data illustrate that the binding of a factor to more-accessible sites, near the end of a nucleosome, allows facilitated binding of additional factors to the center of the nucleosome, thereby overcoming repression from the core histone amino termini. This mechanism may contribute to the binding of multiple factors to complex promoter and enhancer elements in cellular chromatin.
Suppression of albumin enhancer activity by H-ras and AP-1 in hepatocyte cell lines.
Authors: Hu J , Isom H C
Published: October 2015

We demonstrated, using a transient transfection assay, that the albumin enhancer increased the expression of the albumin promoter in a highly differentiated, simian virus 40 (SV40)-immortalized hepatocyte cell line, CWSV1, but was not functional in two ras-transformed cell lines (NR3 and NR4) derived from CWSV1 by stable transfection with the T24ras oncogene. A transient cotransfection assay showed that T24ras and normal c-Ha-ras were each able to inhibit the activity of the albumin enhancer in an immortal hepatocyte cell line. DNase I footprinting and gel mobility shift assays demonstrated that the DNA binding activities specific to the albumin enhancer were not decreased in the ras-transformed cells. ras also did not diminish the expression of HNF1 alpha, C/EBP alpha, HNF3 alpha, HNF3 beta, or HNF3 gamma but did significantly increase AP-1 binding activity. Three AP-1 binding sites were identified within the albumin enhancer, and DNA binding activities specific to these AP-1 sites were induced in the ras-transformed hepatocytes. Subsequent functional assays showed that overexpression of c-jun and c-fos inhibited the activity of the albumin enhancer. Site-directed mutagenesis of the AP-1 binding sites in the albumin enhancer partially abrogated the suppressing effect of ras and c-jun/c-fos on the enhancer. These functional studies therefore supported the results of the structural studies with AP-1. We conclude that the activity of the albumin enhancer is subject to regulation by ras signaling pathways and that the effect of ras on the albumin enhancer activity may be mediated by AP-1.
A new orphan member of the nuclear hormone receptor superfamily that interacts with a subset of retinoic acid response elements.
Authors: Baes M , Gulick T , Choi H S , Martinoli M G , Simha D , Moore D D
Published: October 2015

We have identified and characterized a new orphan member of the nuclear hormone receptor superfamily, called MB67, which is predominantly expressed in liver. MB67 binds and transactivates the retinoic acid response elements that control expression of the retinoic acid receptor beta 2 and alcohol dehydrogenase 3 genes, both of which consist of a direct repeat hexamers related to the consensus AGGTCA, separated by 5 bp. MB67 binds these elements as a heterodimer with the 9-cis-retinoic acid receptor, RXR. However, MB67 does not bind or activate other retinoic acid response elements with alternative hexamer arrangements or any of several other wild-type and synthetic hormone response elements examined. The transactivation of retinoic acid response elements by MB67 is weaker than that conferred by the retinoic acid receptors but does not require the presence of all-trans retinoic acid, 9-cis-retinoic acid, or any exogenously added ligand. We propose that MB67 plays an important role in the complex network of proteins that govern response to retinoic acid and its metabolites.