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M P Kladde

Publications and source records attributed to M P Kladde.

At least 19 recordsLinked to original sources
PubMed2000

Tup1p represses Mcm1p transcriptional activation and chromatin remodeling of an a-cell-specific gene.

In yeast, a number of regulatory proteins expressed only in specific cell types interact with general transcription factors in a combinatorial manner to control expression of cell-type-specific genes. We report a detailed analysis of activation and repression events that occur at the promoter of the a-cell-specific STE6 gene fused to a beta-galactosidase gene in a yeast minichromosome, as well as factors that control the chromatin structure of this promoter both in the minichromosome and in the genomic STE6 locus. Mcm1p results in chromatin remodeling and is responsible for all transcriptional activity from the STE6 promoter in both wild-type a and alpha cells. Matalpha2p cooperates with Tup1p to block both chromatin remodeling and Mcm1p-associated activation. While Matalpha2p represses only Mcm1p, the Tup1p-mediated repression involves both Mcm1p-dependent and -independent mechanisms. Swi/Snf and Gcn5p, required for full induction of the STE6 gene, do not contribute to chromatin remodeling. We suggest that Tup1p can contribute to repression by blocking transcriptional activators, in addition to interacting with transcription machinery and stabilizing chromatin.

ATP-Binding Cassette Transporters↗
PubMed2000

Transcriptional activation of transforming growth factor alpha by estradiol: requirement for both a GC-rich site and an estrogen response element half-site.

17beta-Estradiol (E2) induces transforming growth factor alpha (TGFalpha) gene expression in MCF-7 cells and previous studies have identified a 53 bp (-252 to -200) sequence containing two imperfect estrogen responsive elements (EREs) that contribute to E2 responsiveness. Deletion analysis of the TGFalpha gene promoter in this study identified a second upstream region of the promoter (-623 to -549) that is also E2 responsive. This sequence contains three GC-rich sites and an imperfect ERE half-site, and the specific cis-elements and trans-acting factors were determined by promoter analysis in transient transfection experiments, gel mobility shift assays and in vitro DNA footprinting. The results are consistent with an estrogen receptor alpha (ERalpha)/Sp1 complex interacting with an Sp1(N)(30) ERE half-site ((1/2)) motif in which both ERalpha and Sp1 bind promoter DNA. The ER/Sp1-DNA complex is formed using nuclear extracts from MCF-7 cells but not with recombinant human ERalpha or Sp1 proteins, suggesting that other nuclear factor(s) are required for complex stabilization. The E2-responsive Sp1(N)(x)ERE(1/2) motif identified in the TGFalpha gene promoter has also been characterized in the cathepsin D and heat shock protein 27 gene promoters; however, in the latter two promoters the numbers of intervening nucleotides are 23 and 10 respectively.

Animals↗
PubMed1999

Mechanisms of transcriptional activation of bcl-2 gene expression by 17beta-estradiol in breast cancer cells.

bcl-2 gene expression is induced by 17beta-estradiol (E2) in T47D and MCF-7 human breast cancer cells, and the mechanism of E2 responsiveness was further investigated by analysis of the bcl-2 gene promoter. The -1602 to -1534 distal region (bcl-2j) of the promoter was E2-responsive; however, in gel mobility shift assays, the estrogen receptor alpha (ER(alpha)) did not bind [(32)P]bcl-2j, whereas Sp1 protein formed a retarded band complex. Further analysis demonstrated that the upstream region (-1603 to -1579) of the bcl-2 gene promoter contained two GC/GA-rich sites at -1601 (5'-GGGCTGG-3') and -1588 (3'-GGAGGG-5') that bound Sp1 protein. Subsequent studies confirmed that transactivation by E2 was dependent on ER(alpha)/Sp1 interactions with both GC-rich sites, and this was confirmed by in vitro footprinting. In contrast, a 21-base pair E2-responsive downstream region (-1578 to -1534) did not bind Sp1 or ER(alpha) protein; however, analysis of a complex binding pattern with nuclear extracts showed that ATF-1 and CREB-1 bound to this motif. These data coupled with results of transient transfection studies demonstrated that transcriptional activation by E2 of the -1578 to -1534 region of the bcl-2 gene promoter was dependent on induction of cAMP and subsequent activation through a cAMP response element. Thus, hormone regulation of bcl-2 gene expression in breast cancer cells involves multiple enhancer elements and E2-mediated transactivation does not require direct binding of the estrogen receptor with promoter DNA.

Activating Transcription Factor 1↗
PubMed1998

Cloning, characterization and expression of the gene coding for a cytosine-5-DNA methyltransferase recognizing GpC.

A novel gene encoding a cytosine-5-DNA methyltransferase recognizing the dinucleotide GpC was cloned from Chlorella virus NYs-1 and expressed in both Escherichia coli and Saccharomyces cerevisiae . The gene was sequenced and a predicted polypeptide of 362 amino acids with a molecular weight of 41.903 kDa was identified. The protein contains several amino acid motifs with high similarity to those of other known 5-methylcytosine-forming methyltransferases. In addition, this enzyme, named M. Cvi PI, shares 66% identity and 76% similarity with M. Cvi JI, the only other cytosine-5-DNA methyltransferase cloned from a Chlorella virus. The short, frequently occurring recognition sequence of the new methyltransferase will be very useful for in vivo chromatin structure studies in both yeast and higher organisms.

Amino Acid Sequence↗
PubMed1998

Rapid detection of functional expression of C-5-DNA methyltransferases in yeast.

We have previously employed the cytosine-5-DNA methyltransferase (MTase), M. Sss I, as a probe for chromatin architecture in intact cells. Although M. Sss I offers the highest resolution of any currently available MTase, the difficulty in establishing stable, methylation-positive strains poses a barrier to its general utility as a chromatin probe. We describe a simple screen for M. Sss I-expressing strains that eliminates the purification of PCR products amplified from bisulfite-treated DNA, use of radioisotopes, polyacrylamide sequencing gel electrophoresis, and autoradiography. The high throughput of the method now makes it feasible to introduce M. Sss I into a variety of wild-type and mutant genetic backgrounds.

Base Sequence↗
PubMed1998

Sth1p, a Saccharomyces cerevisiae Snf2p/Swi2p homolog, is an essential ATPase in RSC and differs from Snf/Swi in its interactions with histones and chromatin-associated proteins.

The essential Sth1p is the protein most closely related to the conserved Snf2p/Swi2p in Saccharomyces cerevisiae. Sth1p purified from yeast has a DNA-stimulated ATPase activity required for its function in vivo. The finding that Sth1p is a component of a multiprotein complex capable of ATP-dependent remodeling of the structure of chromatin (RSC) in vitro, suggests that it provides RSC with ATP hydrolysis activity. Three sth1 temperature-sensitive mutations map to the highly conserved ATPase/helicase domain and have cell cycle and non-cell cycle phenotypes, suggesting multiple essential roles for Sth1p. The Sth1p bromodomain is required for wild-type function; deletion mutants lacking portions of this region are thermosensitive and arrest with highly elongated buds and 2C DNA content, indicating perturbation of a unique function. The pleiotropic growth defects of sth1-ts mutants imply a requirement for Sth1p in a general cellular process that affects several metabolic pathways. Significantly, an sth1-ts allele is synthetically sick or lethal with previously identified mutations in histones and chromatin assembly genes that suppress snf/swi, suggesting that RSC interacts differently with chromatin than Snf/Swi. These results provide a framework for understanding the ATP-dependent RSC function in modeling chromatin and its connection to the cell cycle.

Adenosine Triphosphatases↗
PubMed1998

Gal4p-mediated chromatin remodeling depends on binding site position in nucleosomes but does not require DNA replication.

Biochemical studies have demonstrated decreased binding of various proteins to DNA in nucleosome cores as their cognate sites are moved from the edge of the nucleosome to the pseudodyad (center). However, to date no study has addressed whether this structural characteristic of nucleosomes modulates the function of a transcription factor in living cells, where processes of DNA replication and chromatin modification or remodeling could significantly affect factor binding. Using a sensitive, high-resolution methyltransferase assay, we have monitored the ability of Gal4p in vivo to interact with a nucleosome at positions that are known to be inaccessible in nucleosome cores in vitro. Gal4p efficiently bound a single cognate site (UASG) centered at 41 bp from the edge of a positioned nucleosome, perturbing chromatin structure and inducing transcription. DNA binding and chromatin perturbation accompanying this interaction also occurred in the presence of hydroxyurea, indicating that DNA replication is not necessary for Gal4p-mediated nucleosome disruption. These data extend previous studies, which demonstrated DNA replication-independent chromatin remodeling, by showing that a single dimer of Gal4p, without the benefit of cooperative interactions that occur at complex wild-type promoters, is competent for invasion of a preestablished nucleosome. When the UASG was localized at the nucleosomal pseudodyad, relative occupancy by Gal4p, nucleosome disruption, and transcriptional activation were substantially compromised. Therefore, despite the increased nucleosome binding capability of Gal4p in cells, the precise translational position of a factor binding site in one nucleosome in an array can affect the ability of a transcriptional regulator to overcome the repressive influence of chromatin.

Binding Sites↗
PubMed1997

Effects of Sin- versions of histone H4 on yeast chromatin structure and function.

Previous studies have identified single amino acid changes within either histone H3 or H4 (Sin- versions) that allow transcription in the absence of the yeast SWI-SNF complex. The histone H4 mutants are competent for nucleosome assembly in vivo, and the residues that are altered appear to define a discrete domain on the surface of the histone octamer. We have analyzed the effects of the Sin- versions of histone H4 on transcription and chromatin structure in vivo. These histone H4 mutants cause an increased accessibility of nucleosomal DNA to Dam methyltransferase and to micrococcal nuclease. Sin- derivatives of histone H4 also grossly impair the ability of nucleosomes to constrain supercoils in vivo. Nucleosome-mediated repression of the PHO5 gene is severely impaired by these histone H4 mutants; PHO5 expression is derepressed to 31% of the wild-type induced level. In contrast to the induction caused by nucleosome depletion, full PHO5 derepression by Sin- versions of histone H4 requires upstream regulatory elements. In addition, Sin- derivatives of histone H4 do not activate expression from CYC1 or GAL1 promoters that lack UAS elements. We propose that these Sin- mutations alter histone-DNA contact residues that play key roles in restricting the accessibility of nucleosomal DNA to transcription factors.

Chromatin↗
PubMed1996

Direct study of DNA-protein interactions in repressed and active chromatin in living cells.

Current methods for analysis of chromatin architecture are invasive, utilizing chemicals or nucleases that damage DNA, making detection of labile constituents and conclusions about true in vivo structure problematic. We describe a sensitive assay of chromatin structure which is performed in intact, living yeast. The approach utilizes expression of SssI DNA methyltransferase (MTase) in Saccharomyces cerevisiae to provide an order-of-magnitude increase in resolution over previously introduced MTases. Combining this resolution increase with the novel application of a PCR-based, positive chemical display of modified cytosines provides a significant advance in the direct study of DNA-protein interactions in growing cells that enables quantitative footprinting. The validity and efficacy of the strategy are demonstrated in mini-chromosomes, where positioned nucleosomes and a labile, operator-bound repressor are detected. Also, using a heterologous system to study gene activation, we show that in vivo hormone occupancy of the estrogen receptor is required for maximal site-specific DNA binding, whereas, at very high receptor-expression levels, hormone-independent partial occupancy of an estrogen-responsive element was observed. Receptor binding to a palindromic estrogen-responsive element leads to a footprint with strand-specific asymmetry, which is explicable by known structural information.

Chromatin↗
PubMed1994

The non-B-DNA structure of d(CA/TG)n differs from that of Z-DNA.

Chemical probing of two predominantly alternating purine-pyrimidine d(CA/TG)n repeats led us to propose previously that in supercoiled plasmids these elements adopt a non-B-DNA structure distinct from that of Z-DNA formed by d(CG)n sequences. Here, we present further evidence supporting this contention. Reactivity with the conformation-sensitive reagent chloroacetaldehyde, which reacts with unpaired adenines and cytosines, was confined strictly to adenines in the d(CA/TG)n repeat. In contrast, only bases outside the d(CG)n repeat exhibited chloroacetaldehyde reactivity. Two-dimensional gel analysis of topoisomers containing d(CA/TG)n tracts with bases out of strict purine-pyrimidine alteration revealed multiple superhelical-dependent transitions to an alternative left-handed structure. Within individual plasmid molecules, these multiple transitions resulted from the stepwise conversion of contiguous segments of alternating purine-pyrimidine sequence, which are delimited by bases out of alternation, to the full-length alternative conformation. When the left-handed helices increased in length to include more bases out of alternation, the average helical pitch changed substantially to produce a less tightly wound left-handed helix. Overall, these data indicate that d(CA/TG)n tracts adopt a left-handed conformation significantly different from that of the canonical Z-DNA structure of d(CG)n sequences.

Acetaldehyde↗
PubMed1994

Positioned nucleosomes inhibit Dam methylation in vivo.

Escherichia coli Dam DNA methyltransferase can methylate genomic GATC sites when expressed in Saccharomyces cerevisiae. Others have observed changes in the level of methylation at specific sites and suggested that these changes are related to transcriptional state or chromosomal context. To test directly the influence of nucleosome location on the ability of Dam methyltransferase to modify GATC sites in chromatin, we analyzed minichromosomes containing precisely positioned nucleosomes in dam-expressing yeast strains. Levels of methylation at individual GATC sites were rigorously quantified by an oligonucleotide-probing procedure. Within the linker and adjacent 21 bp of nucleosome-associated DNA, GATC sites were highly methylated, whereas methylation was severely inhibited by histone-DNA contacts nearer to the nucleosomal pseudodyad. Other DNA-protein complexes also interfere with Dam methylation. These data are consistent with a model in which nucleosomes exert a repressive influence on the biological functions of DNA by restricting access of trans-acting factors to DNA.

Base Sequence↗
PubMed1993

Interaction between transcription regulatory regions of prolactin chromatin.

The regulation of transcription requires complex interactions between proteins bound to DNA sequences that are often separated by hundreds of base pairs. As demonstrated by a nuclear ligation assay, the distal enhancer and the proximal promoter regions of the rat prolactin gene were found to be juxtaposed. By acting through its receptor bound to the distal enhancer, estrogen stimulated the interaction between the distal and proximal regulatory regions two- to threefold compared to control values. Thus, the chromatin structure of the prolactin gene may facilitate the occurrence of protein-protein interactions between transcription factors bound to widely separated regulatory elements.

Animals↗
PubMed1993

Multiple transitions to non-B-DNA structures occur in the distal regulatory region of the rat prolactin gene.

The developmentally regulated rat prolactin (rPRL) gene presents a promising model system toward understanding the biological role of non-B-DNA structural elements. Two predominantly alternating purine-pyrimidine (APP) (dA-dC)n.(dG-dT)n repeats of 58 and 178 base-pairs flank the (A + T)-rich distal regulatory region. We have characterized several transitions to non-B-DNA structures within this region in negatively supercoiled plasmids by utilizing high resolution chemical probing. Each repeat undergoes a full-length conversion to a novel left-handed helical structure via the stepwise nucleation and propagation of discrete "segments". These segments are delimited by out-of-alternation bases that are susceptible to attack by potassium permanganate and thus appear to be significantly unstacked within the left-handed helices. Moreover, the spatial order of successive right- to left-handed DNA transitions within each repeat exhibits a clear polarity toward the distal regulatory region of the rPRL gene. An additional transition involving the long-range unpairing of (A + T)-rich sequences establishes a directional propagation toward the regulatory region. These data demonstrate a complex series of quasi-independent transitions to non-B-DNA structures that impinge upon a known regulatory control region.

Animals↗
PubMed1989

Transcriptional regulation by estrogen of episomal prolactin gene regulatory elements.

As a first step in defining the role of chromatin structure in steroid-regulated gene transcription, we have established a steroid-responsive minichromosome system that contains the 5' upstream regulatory region of the rat PRL gene (PRL) from -10 to -1960-basepairs fused to the antibiotic resistance gene, Tn5. The hybrid gene was inserted into a bovine papilloma virus (BPV) vector and then transfected into GH3 cells. Southern analysis of total genomic DNA revealed that the PRL-Tn5-BPV DNA existed in the cells as unrearranged episomes or minichromosomes at a level of 25-100 copies/cell. We monitored the estrogen responsiveness of the minichromosome-based PRL regulatory regions by measuring Tn5 mRNA levels. Treatment of GH3 cells for 48 h with 10 nM 17 beta-estradiol (E2) increased Tn5 mRNA levels 3- to 6-fold over those in untreated cells. Concurrently, endogenous PRL mRNA levels were induced 8- to 15-fold. Using nuclear run-on assays, it was found that E2 increased PRL-Tn5 transcription rates approximately 3-fold over those in untreated cells. The induced transcription was mediated through the PRL elements and not through any other cis-acting elements within the minichromosome. The PRL elements that contain a functional enhancer are located 3' downstream of the BPV early gene promoters in the minichromosome. However, there was no detectable effect of the PRL enhancer on BPV early gene transcription. Thus, we have established a minichromosome system containing the transcriptional regulatory elements of the rat PRL gene that responds to E2 in a manner very similar to the endogenous rat PRL gene.

Animals↗