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PubMed2004

The APP intracellular domain forms nuclear multiprotein complexes and regulates the transcription of its own precursor.

The physiological functions of the beta-amyloid precursor protein (APP) may include nuclear signaling. To characterize the role of the APP adaptor proteins Fe65, Jip1b, X11alpha (MINT1) and the chromatin-associated protein Tip60, we analyzed their interactions by confocal microscopy and co-immunoprecipitations. AICD corresponding to S3-cleaved APP bound to Fe65 that transported it to nuclei and docked it to Tip60. These proteins formed AICD-Fe65-Tip60 (AFT) complexes that were concentrated in spherical nuclear spots. gamma-Secretase inhibitors prevented AFT-complex formation with AICD derived from full-length APP. The APP adaptor protein Jip1b also transported AICD to nuclei and docked it to Tip60, but AICD-Jip1b-Tip60 (AJT) complexes had different, speckle-like morphology. By contrast, X11alpha trapped AICD in the cytosol. Induced AICD expression identified the APP-effector genes APP, BACE, Tip60, GSK3beta and KAI1, but not the Notch-effector gene Hes1 as transcriptional targets. These data establish a role for APP in nuclear signaling, and they suggest that therapeutic strategies designed to modulate the cleavage of APP affect AICD-dependent signaling.

Acetyltransferases↗
PubMed1997

Is Cdk7/cyclin H/MAT1 the genuine cdk activating kinase in cycling Xenopus egg extracts?

Formation of active cdk (cyclin dependent kinase)/ cyclin kinases involves phosphorylation of a conserved threonine residue in the T loop of the cdk catalytic-subunit by CAK (Cdk Activating Kinase). CAK was first purified biochemically from higher eukaryotes and identified as a trimeric complex containing a cdk7 catalytic subunit, cyclin H and MAT1 (Ménage à trois), a member of the RING finger family. The same trimeric complex is also part of basal transcription factor TFIIH. In budding yeast, the closest homologs of cdk7 and cyclin H, KIN28 and CCL1, respectively, also associate with TFIIH. However, the KIN28/CCL1 complex does not display CAK activity and a distinct protein kinase able to phosphorylate monomeric CDC28 and GST-cdk2 was recently identified, challenging the identification of cdk7 as the physiological CAK in higher eukaryotes. Here we demonstrate that immunodepletion of cdk7 suppresses CAK activity from cycling Xenopus egg extracts, and arrest them before M-phase. We also show that specific translation of mRNAs encoding Xenopus cdk7 and its associated subunits restores CAK activity in cdk7-immunodepleted Xenopus egg extracts. Hence, the cdk7 complex is necessary and sufficient for activation of cdk-cyclin complexes in cycling Xenopus egg extracts.

Animals↗
PubMed2006

Hepatocyte nuclear factor-4alpha contributes to carbohydrate-induced transcriptional activation of hepatic fatty acid synthase.

Refeeding a carbohydrate-rich meal after a fast produces a co-ordinated induction of key glycolytic and lipogenic genes in the liver. The transcriptional response is mediated by insulin and increased glucose oxidation, and both signals are necessary for optimal induction of FAS (fatty acid synthase). The glucose-regulated component of FAS promoter activation is mediated in part by ChREBP [ChoRE (carbohydrate response element)-binding protein], which binds to a ChoRE between -7300 and -7000 base-pairs in a carbohydrate-dependent manner. Using in vivo footprinting with nuclei from fasted and refed rats, we identify an imperfect DR-1 (direct repeat-1) element between -7110 and -7090 bp that is protected upon carbohydrate refeeding. Electrophoretic mobility-shift assays establish that this DR-1 element binds HNF-4alpha (hepatocyte nuclear factor 4alpha), and chromatin immunoprecipitation establishes that HNF-4alpha binding to this site is increased approx. 3-fold by glucose refeeding. HNF-4alpha transactivates reporter constructs containing the distal FAS promoter in a DR-1-dependent manner, and this DR-1 is required for full glucose induction of the FAS promoter in primary hepatocytes. In addition, a 3-fold knockdown of hepatocyte HNF-4alpha by small interfering RNA produces a corresponding decrease in FAS gene induction by glucose. Co-immunoprecipitation experiments demonstrate a physical interaction between HNF-4alpha and ChREBP in primary hepatocytes, further supporting an important complementary role for HNF-4alpha in glucose-induced activation of FAS transcription. Taken together, these observations establish for the first time that HNF-4alpha functions in vivo through a DR-1 element in the distal FAS promoter to enhance gene transcription following refeeding of glucose to fasted rats. The findings support the broader view that HNF-4alpha is an integral component of the hepatic nutrient sensing system that co-ordinates transcriptional responses to transitions between nutritional states.

Animals↗
PubMed2002

Rhythmic histone acetylation underlies transcription in the mammalian circadian clock.

In the mouse circadian clock, a transcriptional feedback loop is at the centre of the clockwork mechanism. Clock and Bmal1 are essential transcription factors that drive the expression of three period genes (Per1-3) and two cryptochrome genes (Cry1 and Cry2). The Cry proteins feedback to inhibit Clock/Bmal1-mediated transcription by a mechanism that does not alter Clock/Bmal1 binding to DNA. Here we show that transcriptional regulation of the core clock mechanism in mouse liver is accompanied by rhythms in H3 histone acetylation, and that H3 acetylation is a potential target of the inhibitory action of Cry. The promoter regions of the Per1, Per2 and Cry1 genes exhibit circadian rhythms in H3 acetylation and RNA polymerase II binding that are synchronous with the corresponding steady-state messenger RNA rhythms. The histone acetyltransferase p300 precipitates together with Clock in vivo in a time-dependent manner. Moreover, the Cry proteins inhibit a p300-induced increase in Clock/Bmal1-mediated transcription. The delayed timing of the Cry1 mRNA rhythm, relative to the Per rhythms, is due to the coordinated activities of Rev-Erbalpha and Clock/Bmal1, and defines a new mechanism for circadian phase control.

Acetylation↗
PubMed2004

HIF-1alpha induces cell cycle arrest by functionally counteracting Myc.

Hypoxia induces angiogenesis and glycolysis for cell growth and survival, and also leads to growth arrest and apoptosis. HIF-1alpha, a basic helix-loop-helix PAS transcription factor, acts as a master regulator of oxygen homeostasis by upregulating various genes under low oxygen tension. Although genetic studies have indicated the requirement of HIF-1alpha for hypoxia-induced growth arrest and activation of p21(cip1), a key cyclin-dependent kinase inhibitor controlling cell cycle checkpoint, the mechanism underlying p21(cip1) activation has been elusive. Here we demonstrate that HIF-1alpha, even in the absence of hypoxic signal, induces cell cycle arrest by functionally counteracting Myc, thereby derepressing p21(cip1). The HIF-1alpha antagonism is mediated by displacing Myc binding from p21(cip1) promoter. Neither HIF-1alpha transcriptional activity nor its DNA binding is essential for cell cycle arrest, indicating a divergent role for HIF-1alpha. In keeping with its antagonism of Myc, HIF-1alpha also downregulates Myc-activated genes such as hTERT and BRCA1. Hence, we propose that Myc is an integral part of a novel HIF-1alpha pathway, which regulates a distinct group of Myc target genes in response to hypoxia.

Adenoviridae↗
PubMed2002

Esc1, a nuclear periphery protein required for Sir4-based plasmid anchoring and partitioning.

A targeted silencing screen was performed to identify yeast proteins that, when tethered to a telomere, suppress a telomeric silencing defect caused by truncation of Rap1. A previously uncharacterized protein, Esc1 (establishes silent chromatin), was recovered, in addition to well-characterized proteins Rap1, Sir1, and Rad7. Telomeric silencing was slightly decreased in Deltaesc1 mutants, but silencing of the HM loci was unaffected. On the other hand, targeted silencing by various tethered proteins was greatly weakened in Deltaesc1 mutants. Two-hybrid analysis revealed that Esc1 and Sir4 interact via a 34-amino-acid portion of Esc1 (residues 1440 to 1473) and a carboxyl-terminal domain of Sir4 known as PAD4 (residues 950 to 1262). When tethered to DNA, this Sir4 domain confers efficient partitioning to otherwise unstable plasmids and blocks the ability of bound DNA segments to rotate freely in vivo. Here, both phenomena were shown to require ESC1. Sir protein-mediated partitioning of a telomere-based plasmid also required ESC1. Fluorescence microscopy of cells expressing green fluorescent protein (GFP)-Esc1 showed that the protein localized to the nuclear periphery, a region of the nucleus known to be functionally important for silencing. GFP-Esc1 localization, however, was not entirely coincident with telomeres, the nucleolus, or nuclear pore complexes. Our data suggest that Esc1 is a component of a redundant pathway that functions to localize silencing complexes to the nuclear periphery.

Basic Helix-Loop-Helix Proteins↗
PubMed1994

Distinct DNase-I hypersensitive sites are associated with TAL-1 transcription in erythroid and T-cell lines.

The tal-1 gene, frequently activated in human T-cell acute lymphoblastic leukemia (T-ALL), is expressed in the erythroid, megakaryocytic, and mast cell lineages during normal hematopoiesis. To gain further insight into the molecular mechanisms that control tal-1 expression, we investigated tal-1 chromatin structure in erythroid/megakaryocytic cell lines and in T-cell lines either with or without tal-1 rearrangements. Tal-1 transcription was shown to be monoallelic in Jurkat, a T-cell line that expresses tal-1 in the absence of apparent genomic alteration of the locus. Methylation studies indicated that the tal-15' GC-rich region behaves like a CpG island, hypomethylated in normal cells, and methylated de novo on transcriptionally inactive alleles in established cell lines. Five major DNase-I hypersensitive sites (HS) were mapped in the tal-1 locus. HS I, IV, and V were exclusively observed in the erythroid/megakaryocytic cell lines that express tal-1 from the promoters 1a and 1b. HS II was weak in hematopoietic cell lines, absent in Hela, and greatly enhanced in Jurkat, suggesting that this region might be implicated in the cis-activation of tal-1 promoter 1b in this cell line. HS III was weak in HEL and Jurkat, and greatly enhanced in DU528, a T-cell line that bears a t (1;14) and initiates tal-1 transcription within exon 4. These results suggest that distinct regulatory elements are associated with the use of the different tal-1 promoters.

Base Sequence↗
PubMedDate not supplied

Mxi1-0, an alternatively transcribed Mxi1 isoform, is overexpressed in glioblastomas.

The c-Myc transcription factor regulates expression of genes related to cell growth, division, and apoptosis. Mxi1, a member of the Mad family, represses transcription of c-Myc-regulated genes by mediating chromatin condensation via histone deacetylase and the Sin3 corepressor. Mxi1 is a c-Myc antagonist and suppresses cell proliferation in vitro. Here, we describe the identification of Mxi1-0, a novel Mxi1 isoform that is alternatively transcribed from an upstream exon. Mxi1-0 and Mxi1 have different amino-terminal sequences, but share identical Max- and DNA-binding domains. Both isoforms are able to bind Max, to recognize E-box binding sites, and to interact with Sin3. Despite these similarities and in contrast to Mxi1, Mxi1-0 is predominantly localized to the cytoplasm and fails to repress c-Myc-dependent transcription. Although Mxi1-0 and Mxi1 are coexpressed in both human and mouse cells, the relative levels of Mxi1-0 are higher in primary glioblastoma tumors than in normal brain tissue. This variation in the levels of Mxi1-0 and Mxi1 suggests that Mxi1-0 may modulate the Myc-inhibitory activity of Mxi1. The identification of Mxi1-0 as an alternatively transcribed Mxi1 isoform has significant implications for the interpretation of previous Mxi1 studies, particularly those related to the phenotype of the mxi1 knockout mouse.

Amino Acid Sequence↗
PubMed2002

PRCC-TFE3 renal carcinomas: morphologic, immunohistochemical, ultrastructural, and molecular analysis of an entity associated with the t(X;1)(p11.2;q21).

The reappraisal of genetically defined subsets of renal tumors can help to highlight the key pathologic features of specific neoplastic entities. We report the morphologic, immunophenotypic, ultrastructural, and molecular features of 11 renal carcinomas bearing a t(X;1)(p11.2;q21) and/or the resulting PRCC-TFE3 gene fusion. The male/female ratio was 4:7. Ten patients were in the age range of 9-29 years and one was 64 years old (mean 21.3 years, median 15 years). The predominant histologic pattern was nested, with islands of tumor cells compartmentalized by thin-walled capillary vasculature. Minor variations on this pattern yielded solid, acinar, alveolar, and tubular architecture. Papillary architecture was seen in nine cases, usually as a minor component. Neoplastic cells were typically characterized by irregularly shaped nuclei with vesicular chromatin and small nucleoli not visible with a 10x objective, and cytoplasm that ranged from clear to densely granular and eosinophilic. Mitoses were extremely rare; 5 were found in 900 high power fields examined from the 11 neoplasms. The most distinctive immunohistochemical feature of these neoplasms was moderate to intense nuclear labeling for TFE3 protein. These tumors were also consistently immunoreactive for the RCC antigen (10 of 11) and CD10 (9 of 9), whereas cytokeratin and epithelial membrane antigen were negative in four cases and were positive focally in the others. Ultrastructurally, all of the six neoplasms examined showed features consistent with conventional-type (clear cell) renal carcinoma, although two demonstrated distinctive intracisternal microtubules. Both tumors tested contained PRCC-TFE3 fusion transcripts. The differential diagnosis includes conventional-type papillary renal cell carcinoma, conventional-type (clear cell) renal carcinoma, and the ASPL-TFE3 renal carcinomas associated with the t(X;17)(p11.2;q25), with the latter two being morphologically the most similar to the t(X;1) renal carcinomas. Aside from their distinctive clinicopathologic features described here, there is experimental evidence suggesting that these tumors may show differential sensitivity to certain chemotherapeutic agents.

Adolescent↗
PubMed1999

Trithorax and ASH1 interact directly and associate with the trithorax group-responsive bxd region of the Ultrabithorax promoter.

Trithorax (TRX) and ASH1 belong to the trithorax group (trxG) of transcriptional activator proteins, which maintains homeotic gene expression during Drosophila development. TRX and ASH1 are localized on chromosomes and share several homologous domains with other chromatin-associated proteins, including a highly conserved SET domain and PHD fingers. Based on genetic interactions between trx and ash1 and our previous observation that association of the TRX protein with polytene chromosomes is ash1 dependent, we investigated the possibility of a physical linkage between the two proteins. We found that the endogenous TRX and ASH1 proteins coimmunoprecipitate from embryonic extracts and colocalize on salivary gland polytene chromosomes. Furthermore, we demonstrated that TRX and ASH1 bind in vivo to a relatively small (4 kb) bxd subregion of the homeotic gene Ultrabithorax (Ubx), which contains several trx response elements. Analysis of the effects of ash1 mutations on the activity of this regulatory region indicates that it also contains ash1 response element(s). This suggests that ASH1 and TRX act on Ubx in relatively close proximity to each other. Finally, TRX and ASH1 appear to interact directly through their conserved SET domains, based on binding assays in vitro and in yeast and on coimmunoprecipitation assays with embryo extracts. Collectively, these results suggest that TRX and ASH1 are components that interact either within trxG protein complexes or between complexes that act in close proximity on regulatory DNA to maintain Ubx transcription.

Amino Acid Sequence↗
PubMed1998

Cooperative Pho2-Pho4 interactions at the PHO5 promoter are critical for binding of Pho4 to UASp1 and for efficient transactivation by Pho4 at UASp2.

The activation of the PHO5 gene in Saccharomyces cerevisiae in response to phosphate starvation critically depends on two transcriptional activators, the basic helix-loop-helix protein Pho4 and the homeodomain protein Pho2. Pho4 acts through two essential binding sites corresponding to the regulatory elements UASp1 and UASp2. Mutation of either of them results in a 10-fold decrease in promoter activity, and mutation of both sites renders the promoter totally uninducible. The role of Pho4 appears relatively straightforward, but the mechanism of action of Pho2 had remained elusive. By in vitro footprinting, we have recently mapped multiple Pho2 binding sites adjacent to the Pho4 sites, and by mutating them individually or in combination, we now show that each of them contributes to PHO5 promoter activity. Their function is not only to recruit Pho2 to the promoter but to allow cooperative binding of Pho4 together with Pho2. Cooperativity requires DNA binding of Pho2 to its target sites and Pho2-Pho4 interactions. A Pho4 derivative lacking the Pho2 interaction domain is unable to activate the promoter, but testing of UASp1 and UASp2 individually in a minimal CYC1 promoter reveals a striking difference between the two UAS elements. UASp1 is fully inactive, presumably because the Pho4 derivative is not recruited to its binding site. In contrast, UASp2 activates strongly in a Pho2-independent manner. From in vivo footprinting experiments and activity measurements with a promoter variant containing two UASp2 elements, we conclude that at UASp2, Pho2 is mainly required for the ability of Pho4 to transactivate.

Acid Phosphatase↗
PubMed1996

Myc-Max heterodimers activate a DEAD box gene and interact with multiple E box-related sites in vivo.

The c-Myc protein is involved in cell proliferation, differentiation and apoptosis though heterodimerization with Max to form a transcriptionally active sequence-specific DNA binding complex. By means of sequential immunoprecipitation of chromatin using anti-Max and anti-Myc antibodies, we have identified a Myc-regulated gene and genomic sites occupied by Myc-Max in vivo. Four of 27 sites recovered by this procedure corresponded to the highest affinity 'canonical' CACGTG sequence. However, the most common in vivo binding sites belonged to the group of 'non-canonical' E box-related binding sites previously identified by in vitro selection. Several of the genomic fragments isolated contained transcribed sequences, including one, MrDb, encoding an evolutionarily conserved RNA helicase of the DEAD box family. The corresponding mRNA was induced following activation of a Myc-estrogen receptor fusion protein (Myc-ER) in the presence of a protein synthesis inhibitor, consistent with this helicase gene being a direct target of Myc-Max. In addition, as for c-Myc, the expression of MrDb is induced upon proliferative stimulation of primary human fibroblasts as well as B cells and down-regulated during terminal differentiation of HL60 leukemia cells. Our results indicate that Myc-Max heterodimers interact in vivo with a specific set of E box-related DNA sequences and that Myc is likely to activate multiple target genes including a highly conserved DEAD box protein. Therefore, Myc may exert its effects on cell behavior through proteins that affect RNA structure and metabolism.

Amino Acid Sequence↗
PubMed2005

LvGroucho and nuclear beta-catenin functionally compete for Tcf binding to influence activation of the endomesoderm gene regulatory network in the sea urchin embryo.

In the sea urchin embryo, specification of the endomesoderm is accomplished by the activity of a network of regulatory genes in the vegetal hemisphere, called the endomesoderm gene regulatory network (GRN). The activation of this network is mediated primarily through the activity of the Wnt pathway, though details of pathway activation remain unclear. To gain further insight into control of endomesoderm GRN activation, we have identified a sea urchin homologue of the co-repressor Groucho (LvGroucho) that has been shown to antagonize beta-catenin/Tcf activation complexes during Wnt signaling in other systems. Groucho functions by recruiting the histone deacetylase Rpd3 to the DNA template via interaction with site-specific transcription factors, resulting in localized chromatin condensation and transcriptional silencing. Our results show that the LvGroucho protein localizes to all nuclei throughout embryonic development. Interaction assays demonstrate that LvGroucho interacts with Tcf via both the Q and the WD domains of the protein. LvGroucho interacts with Tcf to antagonize the expression of key endomesoderm regulatory genes. Assays demonstrate that LvGroucho and n beta-catenin functionally compete for binding to Tcf as a major mechanism by which the Tcf-control switch is regulated. Functional analysis of the N-terminal AES197 domain of LvGroucho shows that it is sufficient to recapitulate the function of full-length LvGroucho. This finding strongly supports the conclusion that the effects of LvGro overexpression are due primarily to its interactions with Tcf and not other Groucho interacting partners, since Tcf is the only protein present in the sea urchin known to interact with AES197. Because the Q domain is unable to bind Rpd3, it was expected to behave as a dominant negative LvGroucho. Unexpectedly, overexpression of the Q domain gave functional results similar to LvGroucho and the AES197 domain. This is the first evidence for an inherent repressive function for the Q domain alone. Together, our results indicate that LvGroucho functionally competes with beta-catenin for Tcf binding, and this competitive mechanism regulates one of the earliest steps in the initiation of the sea urchin endomesoderm GRN.

Amino Acid Sequence↗
PubMed2001

Role of nuclear WW domains and proline-rich proteins in dinoflagellate transcription.

Dinoflagellates are unique among eukaryotes in their lack of histones and nucleosomes, and permanently condensed chromosomes. These unusual features raise questions as how chromatin condensation and gene expression are achieved. In this study, we investigated nuclear proteins potentially implicated in the regulation of the transcription. Dinap1 is a dinoflagellate nuclear protein that has a WW domain and is synthesized mainly in G1 and S phases of the cell cycle. In this study, we found that Dip1, a proline-rich potential ligand of Dinap1, and DapC, a Dip1 potential ligand, were both present in the nucleus of Crypthecodinium cohnii during the G1 phase. Dip1 contained a PPXY motif, and its domain organization was similar to that of the splicing factor FBP21 in that it possessed one zinc finger and two WW domains. Although DapC has no known homolog, 22 repeats of a PPXPXGX heptapeptide were identified at the N-terminus, and this structure is similar to that of the C-terminal part of the mouse splicing factor SAP62. Dinap1 was co-precipitated with Dip1 and DapC in vitro and in vivo, but despite their nuclear location, these three proteins did not bind directly to DNA. Dinap1 activated up to 40% of the basal transcription activity of C. cohnii in an in vitro assay, whereas DapC inhibited it by 40% and Dip1 had no effect. These dinoflagellate proteins appear to be the subunits of a nuclear complex that may be involved in regulating transcription.

Amino Acid Sequence↗
PubMed2004

Regulation of the human secretin gene is controlled by the combined effects of CpG methylation, Sp1/Sp3 ratio, and the E-box element.

To unravel the mechanisms that regulate the human secretin gene expression, in this study, we have used secretin-expressing (HuTu-80 cells, human duodenal adenocarcinoma) and non-secretin-expressing [PANC-1 (human pancreatic ductile carcinoma) and HepG2 (human hepatocellular carcinoma) cells] cell models for in vitro and in vivo analyses. By transient transfection assays, within the promoter region (-11 to -341 from ATG, relative to the ATG initiation codon), we have initially identified several functional motifs including an E-box and 2 GC-boxes. Results from gel mobility shift and chromatin immunoprecipitation assays confirmed further that NeuroD, E2A, Sp1, and Sp3 bind to these E- and GC-boxes in HuTu-80 cells in vitro and in vivo, whereas only high levels of Sp3 is observed to bind the promoter in HepG2 cells. In addition, overexpression of Sp3 resulted in a dose-dependent repression of the Sp1-mediated transactivation. Collectively, these data suggest that the Sp1/Sp3 ratio is instrumental to controlling secretin gene expression in secretin-producing and non-secretin-producing cells. The functions of GC-box and Sp proteins prompted us to investigate the possible involvement of DNA methylation in regulating this gene. Consistent with this idea, we found a putative CpG island (-336 to 262 from ATG) that overlaps with the human secretin gene promoter. By methylation-specific PCR, all the CpG dinucleo-tides (26 of them) within the CpG island in HuTu-80 cells are unmethylated, whereas all these sites are methylated in PANC-1 and HepG2 cells. The expressions of secretin in PANC-1 and HepG2 cells were subsequently found to be significantly activated by a demethylation agent, 5'-Aza-2' deoxycytidine. Taken together, our data indicate that the human secretin gene is controlled by the in vivo Sp1/Sp3 ratio and the methylation status of the promoter.

5' Flanking Region↗
PubMed2006

Telomestatin-induced telomere uncapping is modulated by POT1 through G-overhang extension in HT1080 human tumor cells.

Telomestatin is a potent G-quadruplex ligand that interacts with the 3' telomeric overhang, leading to its degradation, and induces a delayed senescence and apoptosis of cancer cells. POT1 and TRF2 were recently identified as specific telomere-binding proteins involved in telomere capping and t-loop maintenance and whose interaction with telomeres is modulated by telomestatin. We show here that the treatment of HT1080 human tumor cells by telomestatin induces a rapid decrease of the telomeric G-overhang and of the double-stranded telomeric repeats. Telomestatin treatment also provokes a strong decrease of POT1 and TRF2 from their telomere sites, suggesting that the ligand triggers the uncapping of the telomere ends. The effect of the ligand is associated with an increase of the gamma-H2AX foci, one part of them colocalizing at telomeres, thus indicating the occurrence of a DNA damage response at the telomere, but also the presence of additional DNA targets for telomestatin. Interestingly, the expression of GFP-POT1 in HT1080 cells increases both telomere and G-overhang length. As compared with HT1080 cells, HT1080GFP-POT1 cells presented a resistance to telomestatin treatment characterized by a protection to the telomestatin-induced growth inhibition and the G-overhang shortening. This protection is related to the initial G-overhang length rather than to its degradation rate and is overcome by increased telomestatin concentration. Altogether these results suggest that telomestatin induced a telomere dysfunction in which G-overhang length and POT1 level are important factors but also suggest the presence of additional DNA sites of action for the ligand.

Base Sequence↗
PubMed2004

Predicting interactions of winged-helix transcription factors with DNA.

Determining protein-DNA interactions is important for understanding gene regulation, DNA repair and chromatin structure. Unfortunately, the structures of DNA-bound complexes are often difficult to obtain experimentally, so the development of computational methods that provide good models of these complexes would be valuable. Here, we present a rigid-body docking approach using the computer program DOT. DOT performs a complete, six-dimensional search of all orientations for two rigid molecules and calculates the interaction energy as the sum of electrostatic and van der Waals terms. DOT was applied to three winged-helix transcription factors that share similar DNA-binding structural motifs but bind DNA in different ways. Docking with linear B-form DNA models accomplished several objectives; it (1) distinguished the different ways the transcription factors bind DNA, (2) identified each protein's DNA-binding site and the DNA orientation at the site and (3) gave at least one solution among the three best-ranked that shows the protein side chain-DNA base interactions responsible for recognition. Furthermore, the ensemble of top-ranked, docked linear B-DNA fragments indicated the DNA bending induced upon protein binding. Docking linear B-DNA to structures of the transcription factor FadR suggests that the allosteric, conformational change induced upon effector binding results in loss of the ability to bend DNA as well as loss of sequence-specific interactions with DNA. The electrostatic energy term calculated by DOT is comparable to the electrostatic binding energy calculated by Poisson-Boltzmann methods. Our results show rigid-body docking that includes a rigorous treatment of the electrostatic interaction energy can be effective in predicting protein-DNA interactions.

Bacterial Proteins↗
PubMed2000

HMG-I/Y, a new c-Myc target gene and potential oncogene.

The HMG-I/Y gene encodes the HMG-I and HMG-Y proteins, which function as architectural chromatin binding proteins important in the transcriptional regulation of several genes. Although increased expression of the HMG-I/Y proteins is associated with cellular proliferation, neoplastic transformation, and several human cancers, the role of these proteins in the pathogenesis of malignancy remains unclear. To better understand the role of these proteins in cell growth and transformation, we have been studying the regulation and function of HMG-I/Y. The HMG-I/Y promoter was cloned, sequenced, and subjected to mutagenesis analysis. A c-Myc-Max consensus DNA binding site was identified as an element important in the serum stimulation of HMG-I/Y. The oncoprotein c-Myc and its protein partner Max bind to this site in vitro and activate transcription in transfection experiments. HMG-I/Y expression is stimulated by c-Myc in a Myc-estradiol receptor cell line in the presence of the protein synthesis inhibitor cycloheximide, indicating that HMG-I/Y is a direct c-Myc target gene. HMG-I/Y induction is decreased in Myc-deficient fibroblasts. HMG-I/Y protein expression is also increased in Burkitt's lymphoma cell lines, which are known to have increased c-Myc protein. Like Myc, increased expression of HMG-I protein leads to the neoplastic transformation of both Rat 1a fibroblasts and CB33 cells. In addition, Rat 1a cells overexpressing HMG-I protein form tumors in nude mice. Decreasing HMG-I/Y proteins using an antisense construct abrogates transformation in Burkitt's lymphoma cells. These findings indicate that HMG-I/Y is a c-Myc target gene involved in neoplastic transformation and a member of a new class of potential oncogenes.

Animals↗