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PubMed2004

The doubletime and CKII kinases collaborate to potentiate Drosophila PER transcriptional repressor activity.

In all genetically studied model organisms, a negative feedback loop of gene expression contributes to the circadian rhythm mechanism. In the Drosophila system, it has been proposed that the delay between the synthesis and function of clock proteins is due to phosphorylation-regulated nuclear entry. To test this hypothesis, we assayed the relationship between PER phosphorylation, nuclear localization, and transcriptional repression activity in cultured S2 cells. The results indicate that the two putative PER kinases DBT and CKII work together to phosphorylate PER and increase repression activity. Experiments combining kinase inhibition with inhibition of PER nuclear export suggest that phosphorylation directly affects PER repression activity and that PER nuclear localization is an indirect consequence of the association of active PER with DNA or chromatin. This interpretation suggests further that the circadian regulation of PER nuclear localization in flies reflects changes in PER transcriptional activity rather than in PER nuclear import or export activity.

Active Transport, Cell Nucleus↗
PubMed2004

Upstream stimulatory factors, USF1 and USF2, bind to the human haem oxygenase-1 proximal promoter in vivo and regulate its transcription.

The human HO-1 (haem oxygenase-1) gene encodes a microsomal enzyme responsible for the breakdown of haem, and is also cytoprotective in response to various cellular insults. HO-1 transcription is induced by a vast array of compounds including, but certainly not limited to, haem and heavy metals such as cadmium. In the present study, we show that upstream stimulatory factors, USF1 and USF2, ubiquitous proteins belonging to the basic helix-loop-helix-leucine zipper family of transcription factors, constitutively bind to the class B E-box located in the proximal promoter of the human HO-1 gene and are responsible for the enhancement of HO-1 gene transcription in human renal proximal tubular epithelial cells. Dimethylsulphate in vivo footprinting studies have identified three protected guanine residues in the E-box of the HO-1 proximal promoter. One of these guanine contact points is essential for USF binding, and when mutated mimics a deletion mutation of the entire E-box palindrome sequence encompassing all three guanine contact points. Binding of USF1 and USF2 to the HO-1 E-box was confirmed by chromatin immunoprecipitation and gel-shift assays. Furthermore, we show that overexpression of USF1 or USF2 enhances the basal expression of HO-1 and that expression of a USF dominant negative form reduces its expression. These results demonstrate for the first time that USF proteins bind to the human HO-1 promoter in vivo and are required for high-level expression of HO-1 by haem and cadmium in human renal epithelial cells.

Animals↗
PubMed2003

Human papillomavirus E6 and Myc proteins associate in vivo and bind to and cooperatively activate the telomerase reverse transcriptase promoter.

The papillomavirus E6 protein binds and directs the ubiquitin-dependent degradation of the p53 tumor suppressor protein. Independent of this p53-degradative function, however, E6 induces cellular telomerase activity. This increase in enzyme activity reflects E6-enhanced transcription of the human telomerase reverse transcriptase (hTERT) catalytic subunit, but the molecular basis for this transactivation is unknown. In the present study, we demonstrate that E6/Myc interactions regulate hTERT gene expression. Mad protein, a specific antagonist of Myc, repressed E6-mediated transactivation of the hTERT promoter and this repression was relieved by Myc overexpression. The proximal Myc/ Max-binding element (E-box) in the hTERT promoter was the major determinant of both E6 and Myc responsiveness in keratinocytes. E6 did not alter Myc protein expression or Myc/Max association, and the induction of hTERT by Myc/E6 was independent of Myc phosphorylation at Thr-58/Ser-62 within the transactivation domain. However, immunoprecipitation studies demonstrated that endogenous Myc protein coprecipitated with E6 protein and chromatin immunoprecipitation analyses demonstrated that both E6 and Myc proteins bound to a minimal 295-bp hTERT promoter. Only the "high-risk" E6 proteins bound to the hTERT promoter, consistent with their preferential ability to induce telomerase. The observation that E6 associates with Myc complexes and activates a Myc-responsive gene identifies a mechanism by which this oncogene can modulate cell proliferation and differentiation.

Basic Helix-Loop-Helix Leucine Zipper Transcriptio↗
PubMed2000

The Myc/Max/Mad network and the transcriptional control of cell behavior.

The Myc/Max/Mad network comprises a group of transcription factors whose distinct interactions result in gene-specific transcriptional activation or repression. A great deal of research indicates that the functions of the network play roles in cell proliferation, differentiation, and death. In this review we focus on the Myc and Mad protein families and attempt to relate their biological functions to their transcriptional activities and gene targets. Both Myc and Mad, as well as the more recently described Mnt and Mga proteins, form heterodimers with Max, permitting binding to specific DNA sequences. These DNA-bound heterodimers recruit coactivator or corepressor complexes that generate alterations in chromatin structure, which in turn modulate transcription. Initial identification of target genes suggests that the network regulates genes involved in the cell cycle, growth, life span, and morphology. Because Myc and Mad proteins are expressed in response to diverse signaling pathways, the network can be viewed as a functional module which acts to convert environmental signals into specific gene-regulatory programs.

Animals↗
PubMed2004

Upstream stimulatory factor but not c-Myc enhances transcription of the human polymeric immunoglobulin receptor gene.

Secretory antibodies protect mucosal surfaces from ingested, inhaled and sexually transmitted pathogens. The polymeric immunoglobulin receptor (pIgR) transports antibodies across mucosal epithelia into external secretions. We and others have identified a region of the human polymeric immunoglobulin receptor gene (locus PIGR) that is sufficient for basal transcriptional activity. An E-Box motif, which binds transcription factors of the basic helix-loop-helix/leucine zipper (bHLH/zip) family, was identified as a major regulatory element in the PIGR gene promoter. Transient transfection of PIGR promoter reporter plasmids in intestinal epithelial cell (IEC) lines suggested that the transcription factors upstream stimulatory factor (USF) and c-Myc may exert opposing effects on PIGR promoter activity. Mutations within and flanking the E-Box that favored USF binding enhanced promoter activity, while mutations that favored c-Myc binding reduced promoter activity. Ectopic expression of USF1 or USF2 enhanced PIGR promoter activity, while exogenous c-Myc did not. Electrophoretic mobility shift assays (EMSA) demonstrated that USF1 and USF2 bound to the E-Box motif as homo- and heterodimers. Chromatin immunoprecipitation (ChIP) demonstrated that USF proteins bind the PIGR promoter in vivo, which is enriched in acetylated histones. E-Box motifs are commonly observed in promoters of genes that are highly expressed in the human colon. Genes that are down-regulated in colorectal cancer, including PIGR, frequently have non-canonical E-Boxes, while genes that are up-regulated in colorectal cancer generally have canonical E-Boxes. The results of our experiments may shed light on the mechanisms of dysregulated expression of pIgR in inflammatory bowel disease and colorectal cancer, diseases associated with aberrant expression of c-Myc.

Animals↗
PubMed1994

DNA bending creates favored sites for retroviral integration: an explanation for preferred insertion sites in nucleosomes.

The choice of retroviral integration sites is strongly influenced by chromatin: integration in vitro occurs more efficiently into nucleosomal DNA than into naked DNA, and a characteristic pattern of preferred insertion sites with a 10 bp periodicity is observed at the outer face of the nucleosomal DNA. At least three features of nucleosomal DNA could be responsible for the creation of these favored sites: the presence of histones, attachment of the DNA to a protein surface, and DNA bending. To test each of these possibilities, we studied integration in vitro with human immunodeficiency virus and murine leukemia virus integrases into four model targets that mimic features of nucleosomal DNA: (i) catabolite activator protein-DNA complexes; (ii) lac repressor-operator complexes; (iii) lac repressor-induced loops; and (iv) intrinsically bent A-tract DNA. We found that bending of the target DNA can create favored integration sites at the outer face of the helix, irrespective of whether the bent DNA is attached to a protein surface. Our findings offer an explanation for the preferred usage of nucleosomes as integration targets. In addition, they suggest that bending of the target DNA might be an intrinsic feature of the integration reaction.

Base Sequence↗
PubMed2003

Transcription factor as a topological homeostat.

Abundant prokaryotic chromatin architectural proteins often function also as global transcriptional regulators. In addition, some of this class of proteins modulate the activity of cellular topoisomerases and hence, the superhelical density of DNA. The relationships between the global effect of these proteins on DNA topology and their local effects exerted on particular promoter regions remain largely unexplored. One of the best-characterised examples of this class of proteins is the pleiotropic regulator of metabolism FIS, which reduces the activity of DNA gyrase and counteracts the increase of the overall superhelicity of DNA during early exponential growth phase. Binding of FIS to supercoiled DNA molecules in vitro leads to the formation of branched structures and consequent multiplication of apical loops, whereas on bending the upstream regions of stable RNA promoters FIS acts as a topological homeostat maintaining high local levels of supercoiling required for promoter activity. We argue that the coordinated effects of FIS on the global and local DNA architecture optimise gene expression by channelling the free energy of negative supercoiling to specific, biologically relevant sites.

Factor For Inversion Stimulation Protein↗
PubMed1991

Working with the confocal scanning UV-laser microscope: specific DNA localization at high sensitivity and multiple-parameter fluorescence.

The use of fast-staining DNA-specific dyes such as DAPI or Hoechst 33342/33258 has been a major problem for confocal scanning laser microscopy (CSLM) studies of intranuclear chromatin organization. Moreover, the availability of a confocal ultraviolet scanning laser microscope configuration, which allows an excitation at wavelengths of 364 nm as well as 488, 514 and 543 nm, is a prerequisite for single as well as multiple fluorescence parameter studies, especially if these studies are concerned with the precise localization of intranuclear signals. Here we report the characteristics and application of a CSLM, which was adapted for UV-excitation and therefore enables comparison of the spatial distribution of several types of signals within one preparation. In addition to multiple-parameter studies, we have also investigated the sensitivity of the system with regard to the identification of the double-stranded DNA of lampbrush chromosome loops in germinal vesicles of amphibian oocytes.

Animals↗
PubMed1999

Effects of chromosomal integration site upon p53 interactions with DNA consensus sequence homologies.

In the present study, we report that, despite the presence of one perfect p53 consensus sequence homology (designated SCL CS) and four half-sites within the 3'-untranslated region of the stem cell leukemia (SCL) gene, the native endogenous gene is not regulated by p53. We employ a tet-repressible system to show that, under conditions in which the WAF1 mRNA steady-state level is upregulated fourfold by p53, the SCL mRNA level is not altered. In a previous report, we demonstrated that p53 interactions with the SCL CS can upregulate downstream reporter gene activity 43-fold in transient reporter assays. This disparity prompted us to explore the differences between p53 regulation of SCL CS activity in organized (chromosomally integrated) and disorganized (non-replicating episomal plasmid) chromatin. We show that p53 can increase (between 3-80-fold), decrease (between 5-33-fold) or have no effect upon transactivation of an SCL CS/reporter fusion gene depending upon chromosomal integration site. Most studies used to characterize p53 binding sites employ transient transfection assays. Our results suggest that characterization of consensus sequence homologies by assay of transiently transfected cells may be inaccurate.

3' Untranslated Regions↗
PubMed2006

Antagonistic regulation of beta-globin gene expression by helix-loop-helix proteins USF and TFII-I.

The human beta-globin genes are expressed in a developmental stage-specific manner in erythroid cells. Gene-proximal cis-regulatory DNA elements and interacting proteins restrict the expression of the genes to the embryonic, fetal, or adult stage of erythropoiesis. In addition, the relative order of the genes with respect to the locus control region contributes to the temporal regulation of the genes. We have previously shown that transcription factors TFII-I and USF interact with the beta-globin promoter in erythroid cells. Herein we demonstrate that reducing the activity of USF decreased beta-globin gene expression, while diminishing TFII-I activity increased beta-globin gene expression in erythroid cell lines. Furthermore, a reduction of USF activity resulted in a significant decrease in acetylated H3, RNA polymerase II, and cofactor recruitment to the locus control region and to the adult beta-globin gene. The data suggest that TFII-I and USF regulate chromatin structure accessibility and recruitment of transcription complexes in the beta-globin gene locus and play important roles in restricting beta-globin gene expression to the adult stage of erythropoiesis.

Animals↗
PubMedDate not supplied

Structure of DNase I at 2.0 A resolution suggests a mechanism for binding to and cutting DNA.

Bovine pancreatic deoxyribonuclease I (DNase I), an endonuclease that degrades double-stranded DNA in a nonspecific but sequence-dependent manner, has been used as a biochemical tool in various reactions, in particular as a probe for the structure of chromatin and for the helical periodicity of DNA on the nucleosome and in solution. Limited digestion by DNase I, termed DNase I 'footprinting', is routinely used to detect protected regions in DNA-protein complexes. Recently, we have solved the three-dimensional structure of this glycoprotein (relative molecular mass 30,400) by X-ray structure analysis at 2.5 A resolution and have subsequently refined it crystallographically at 2.0 A. Based on the refined structure and the binding of Ca2+-thymidine 3',5'-diphosphate (Ca-pTp) at the active site, we propose a mechanism of action and present a model for the interaction of DNase I with double-stranded DNA that involves the binding of an exposed loop region in the minor groove of B-DNA and electrostatic interactions of phosphates from both strands with arginine and lysine residues on either side of this loop. We explain DNase I cleavage patterns in terms of this model and discuss the consequences of the extended DNase I-DNA contact region for the interpretation of DNase I footprinting results.

Binding Sites↗
PubMed2004

Identification of a commonly amplified 4.3 Mb region with overexpression of C8FW, but not MYC in MYC-containing double minutes in myeloid malignancies.

Double minutes (dmin), the cytogenetic hallmark of genomic amplification, are found in approximately 1% of karyotypically abnormal acute myeloid leukemias (AML) and myelodysplastic syndromes (MDS). The MYC gene at 8q24 has been reported to be amplified in the majority of the cases, and generally it has been assumed that MYC is the target gene. However, only a few studies have focused on the extent of the amplicon or on the expression patterns of the amplified genes. We have studied six cases (five AML and one MDS) with MYC-containing dmin. Detailed fluorescence in situ hybridization analyses identified a common 4.3 Mb amplicon, with clustered proximal and distal breakpoints, harboring eight known genes (C8FW, NSE2, POU5FLC20, MYC, PVT1, AK093424, MGC27434 and MLZE). The corresponding region was deleted in one of the chromosome 8 homologues in five of the six cases, suggesting that the dmin originated through extra replication (or loop-formation)--excision--amplification. Northern blot analysis revealed that MYC was not overexpressed. Instead, the C8FW gene, encoding a phosphoprotein regulated by mitogenic pathways, displayed increased expression. These results exclude MYC as the target gene and indicate that overexpression of C8FW may be the functionally important consequence of 8q24 amplicons in AML and MDS.

Acute Disease↗
PubMed1989

Fine-structural aspects of bromodeoxyuridine incorporation in sister chromatid differentiation and replication banding.

The structure of harlequin-stained chromosomes following substitution with low levels of 5-bromodeoxyuridine (BrdUrd) over two cell cycles and high levels over the last part of one cycle (replication banding) was studied in Chinese hamster ovary (CHO) cells. By using correlative light (LM) and scanning electron microscopy (SEM), it was shown that the effects of both the ultraviolet light (u.v.) and hot SSC treatment steps of the harlequin staining procedure were necessary to obtain sister-chromatid differentiation (SCD) or replication banding. u.v. treatment alone resulted in dark Giemsa staining of both chromatids with SEM morphology of short compact protuberances and an overall flattened smooth appearance in both the unsubstituted and BrdUrd-substituted chromatids, a morphology essentially similar to that of untreated chromosomes. SSC alone on the other hand resulted in dark-staining chromatids with an SEM morphology of raised, loosely packed loops of fibres in both types of chromatids. u.v. and SSC treatment together resulted in differentiation, with dark-staining unifilarly (TB) chromatids in the LM corresponding to raised loosely packed loops in the SEM and pale bifilarly (BB) chromatids corresponding to the smooth compact flattened SEM appearance. Where the BrdUrd-substituted strand became the template (BT), or when the nascent strand TB contained high levels of BrdUrd substitution in replication banding, the chromatid stained pale and showed the compact smooth appearance in the SEM. The Giemsa staining ability and ultrastructural morphology of harlequin staining is discussed with respect to putative DNA loss and also in terms of preferential protein-protein, protein-DNA cross-linkage in BrdUrd-containing DNA. These changes are also compared with the ultrastructural morphology observed after other banding methods, where deterioration of protein and DNA-protein interaction resulting in aggregation of chromatin fibres appears to be the major mechanism.

Animals↗
PubMed2006

Regulation of Th2 differentiation and Il4 locus accessibility.

Helper T cells coordinate immune responses through the production of cytokines. Th2 cells express the closely linked Il4, Il13, and Il5 cytokine genes, whereas these same genes are silenced in the Th1 lineage. The Th1/Th2 lineage choice has become a textbook example for the regulation of cell differentiation, and recent discoveries have further refined and expanded our understanding of how Th2 differentiation is initiated and reinforced by signals from antigen-presenting cells and cytokine-driven feedback loops. Epigenetic changes that stabilize the active or silent state of the Il4 locus in differentiating helper T cells have been a major focus of recent research. Overall, the field is progressing toward an integrated model of the signaling and transcription factor networks, cis-regulatory elements, epigenetic modifications, and RNA interference mechanisms that converge to determine the lineage fate and gene expression patterns of differentiating helper T cells.

Animals↗
PubMed2006

A novel activating function of c-Src and Stat3 on HGF transcription in mammary carcinoma cells.

In the normal breast, hepatocyte growth factor (HGF) is primarily expressed by stromal cells, and stimulates in a paracrine manner epithelial cells expressing the HGF receptor (Met). In invasive human breast carcinomas, HGF and Met are frequently overexpressed, possibly establishing an autocrine HGF/Met loop that promotes tumour cell invasion. However, the mechanisms leading to autocrine HGF expression in carcinoma cells are not known. We previously demonstrated a cooperative effect between c-Src and Stat3 in the activation of HGF transcription in mammary carcinoma cells. The present report defines a novel Stat3 consensus site at nt -95 in the HGF promoter that is highly conserved in human and mouse, and is required for c-Src and Stat3 to activate HGF transcription in breast epithelial cells. DNA-protein binding studies demonstrated high affinity binding of a Stat3-containing complex to the nt -95 site. Endogenous Stat3 binding to this region of the HGF promoter in carcinoma cells expressing HGF was demonstrated using a chromatin immunoprecipitation assay. In addition, coexpression of Stat3 and activated c-Src caused increased expression of endogenous HGF mRNA and protein and marked cell scattering in breast epithelial cells. Our results delineate a novel c-Src/Stat3-dependent mechanism that regulates HGF promoter activity, and is linked to transformation of mammary epithelial cells.

Animals↗
PubMed2004

Occupancy and synergistic activation of the FMR1 promoter by Nrf-1 and Sp1 in vivo.

Fragile X syndrome is due to mutation of the FMR1 gene. The most common mutation is an expansion of a CGG repeat in the 5' UTR that triggers dense DNA methylation and formation of a heterochromatin-like structure which lead to transcriptional silencing. In vitro experiments have identified several transcription factors, including Sp1, Nrf-1 and USF1/2, as potential regulators of normal FMR1 promoter activity. Using CpG methylation-deficient Drosophila cells, we demonstrate in vivo that Nrf-1 and Sp1 are strong, synergistic activators of an unmethylated human FMR1-driven reporter, while USF1/2 and Max repress this activation. In addition, analyses of transcription factor activity upon DNA methylation of the reporter show that Sp1 activity was largely intact when the promoter was densely methylated, but Nrf-1 transactivation was very sensitive to dense methylation. Notably, Nrf-1 transactivation was relatively insensitive to methylation of cytosines only at its binding site. FMR1 reporter activity is also reduced in HeLa cells after expression of a short interfering RNA directed against endogenous Nrf-1. Using chromatin immunoprecipitation, we demonstrate directly that Sp1 and Nrf-1 occupy the human FMR1 promoter in vivo and these interactions are disrupted in fragile X patient cells. In addition, we discover that Max resides at the FMR1 promoter and show that USF1/2 but not c-Myc are present at endogenous FMR1. These findings provide the first direct in vivo evidence identifying the specific transcription factors that regulate FMR1.

Animals↗
PubMed1987

Intranuclear localization of UV-induced DNA repair in human VA13 cells.

We have investigated the intranuclear localization of DNA-repair synthesis in G1-phase VA13 human cells. Ultraviolet-irradiated cells were permitted to perform unscheduled DNA synthesis in 3H-thymidine (3H-TdR) and then extracted with nonionic detergent and 2 M NaCl to produce nucleoids in which residual nuclear matrix was surrounded by an extended halo of DNA loops. Autoradiographic analysis of these structures permitted discrimination of DNA repair between the matrix and halo regions. Repair label in nucleoids prepared from cells after exposure to fluences of 2.5-30 J/m2 exhibited a dose-dependent association with the nuclear matrix, which ranged from 80% after 2.5 J/m2 to 50% after 30 J/m2. These results support the view that DNA repair is a nuclear matrix-associated process. This conclusion is in agreement with our preliminary study (Harless et al., 1983) and the results of McCready and Cook (1984) but contrasts with that of Mullenders et al. (1983). Questions concerning the differing experimental designs and their potential effects on the localization of DNA repair are discussed. The implications of these results to previous attempts to isolate chromatin factors associated with DNA repair are also considered.

Cell Line↗
PubMed1999

Regulation of HIV-1 transcription.

Human immunodeficiency virus type-1 (HIV-1) is a highly pathogenic lentivirus that requires transcription of its provirus genome for completion of the viral life cycle and the production of progeny virions. Since the first genetic analysis of HIV-1 in 1985, much has been learned about the transcriptional regulation of the HIV-1 genome in infected cells. It has been demonstrated that HIV-1 transcription depends on a varied and complex interaction of host cell transcription factors with the viral long terminal repeat (LTR) promoter. The regulatory elements within the LTR interact with constitutive and inducible transcription factors to direct the assembly of a stable transcription complex that stimulates multiple rounds of transcription by RNA polymerase II (RNAPII). However, the majority of these transcripts terminate prematurely in the absence of the virally encoded trans-activator protein Tat, which stimulates HIV-1 transcription elongation by interacting with a stem-loop RNA element (TAR) formed at the extreme 5' end of all viral transcripts. The Tat-TAR interaction recruits a cellular kinase into the initiation-elongation complex that alters the elongation properties of RNAPII during its transit through TAR. This review summarizes our current knowledge and understanding of the regulation of HIV-1 transcription in infected cells and highlights the important contributions human lentivirus gene regulation has made to our general understanding of the transcription process.

Base Sequence↗