Mapping of nucleosome positions.
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Cell Fractionation↗
Biomedical subjects
F Thoma
Publications and source records attributed to F Thoma.
Reciprocal interferences between nucleosomal organization and transcriptional activity of the yeast SNR6 gene.
Recent work has demonstrated a repressive effect of chromatin on the transcription of the yeast SNR6 gene in vitro. Here, we show the relations between chromatin structure and transcriptional activity of this gene in vivo. Analysis of the SNR6 locus by micrococcal nuclease digestion showed a protection of the TATA box, nuclease-sensitive sites around the A and B blocks, and arrays of positioned nucleosomes in the flanking regions. Analysis of a transcriptionally silent SNR6 mutant containing a 2-bp deletion in the B block showed a loss of TATA-protection and rearrangement or destabilization of nucleosomes in the flanking regions. Hence, SNR6 organizes the chromatin structure in the whole region in a manner dependent on its transcriptional state. Transcriptional analysis was performed by use of maxi-gene SNR6 constructs introduced into histone-mutated strains. Chromatin disruption induced by histone H4 depletion stimulated the transcription of promoter-deficient, but not of wild-type SNR6 genes, revealing a competition between the formation of nucleosomes and the assembly of Pol III transcription complexes that was much in favor of transcription factors. On the other hand, amino-terminal mutations in histone H3 or H4 had no effect (H4) or only a moderate stimulatory effect (H3) on the transcription of promoter-deficient SNR6 genes.
Base Sequence↗
Transcription through the yeast origin of replication ARS1 ends at the ABFI binding site and affects extrachromosomal maintenance of minichromosomes.
When the function of origins of replication in yeast was compromised by placing ARS sequences downstream of strong promoters, ARS activity might have been affected either by transcription or by an altered chromatin configuration induced by the construct. To distinguish between these possibilities, derivatives of the yeast TRP1ARS1 minichromosome were constructed that contained either the DED1 or the PET56 promoter firing against ARS1 (DEDARS and PETARS constructs). PETARS constructs transformed yeast at high frequencies and were maintained as minichromosomes consistent with efficient ARS1 function, but DEDARS constructs transformed at low frequencies and had to be rescued as minichromosomes by insertion of a second ARS (H4-ARS). Chromatin analysis revealed that the ARS1 regions in PETARS and H4-DEDARS constructs were indistinguishable from the ARS1 region of the host TRP1ARS1 circle showing a nuclease sensitive region flanked by a nucleosome. However, RNA-analysis in the ARS region showed high and low levels of transcripts in H4-DEDARS and PETARS, respectively. Transcription elongated through the A, B1, and B2 elements and ended in B3, the binding site for ABFI. We conclude that transcription through ARS1 and not an altered chromatin structure affected ARS activity in these constructs.
Binding Sites↗
Histone H1 expressed in Saccharomyces cerevisiae binds to chromatin and affects survival, growth, transcription, and plasmid stability but does not change nucleosomal spacing.
Histone H1 is proposed to serve a structural role in nucleosomes and chromatin fibers, to affect the spacing of nucleosomes, and to act as a general repressor of transcription. To test these hypotheses, a gene coding for a sea urchin histone H1 was expressed from the inducible GAL1 promoter in Saccharomyces cerevisiae by use of a YEp vector for high expression levels (strain YCL7) and a centromere vector for low expression levels (strain YCL1). The H1 protein was identified by its inducibility in galactose, its apparent molecular weight, and its solubility in 5% perchloric acid. When YCL7 was shifted from glucose to galactose for more than 40 h to achieve maximal levels of H1, H1 could be copurified in approximately stoichiometric amounts with core histones of Nonidet P-40-washed nuclei and with soluble chromatin fractionated on sucrose gradients. While S. cerevisiae tolerated the expression of low levels of H1 in YCL1 without an obvious phenotype, the expression of high levels of H1 correlated with greatly reduced survival, inhibition of growth, and increased plasmid loss but no obvious change in the nucleosomal repeat length. After an initial induction, RNA levels for GAL1 and H1 were drastically reduced, suggesting that H1 acts by the repression of galactose-induced genes. Similar effects, but to a lower extent, were observed when the C-terminal tail of H1 was expressed.
Animals↗
Chromatin transitions during activation and repression of galactose-regulated genes in yeast.
To study the fate of nucleosomes during transcription, a yeast gene 'GAL-URARIB' was constructed which is tightly regulated by the GAL1 promoter and shows in its inactive state a series of positioned nucleosomes that are sensitive for monitoring structural changes by micrococcal nuclease. Upon transcriptional activation, nucleosome positions were lost, but a residual nucleosomal repeat with an altered repeat length and no changes in psoralen accessibility measured by a band shift assay indicated that nucleosomes were present but rearranged on the transcribed gene. When chromatin was prepared 10 or 50 min after glucose repression, nucleosomes were repositioned in a large fraction of the population by a rapid process which most likely did not depend on histone synthesis or DNA replication. However, complete regeneration of the inactive structure and repeat length was observed after one cell generation (2.5 h) suggesting that in this step some missing histones were replaced. The results are consistent with a local dissociation of nucleosomes at the site of the polymerase followed by a rapid reassembly into nucleosomes behind it. The data are further supported by analysis of the chromosomal GAL1, GAL7 and GAL10 genes.
Blotting, Northern↗
Transcription, nucleosome stability, and DNA repair in a yeast minichromosome.
The template strand of the URA3 gene in the minichromosome YRpTRURAP is repaired of UV-induced cyclobutyl pyrimidine dimers (PD) much more efficiently than the nontemplate strand in growth-arrested Saccharomyces cerevisiae cells (Smerdon, M. J., and Thoma, F. (1990) Cell 61, 675). However, other regions of the plasmid are also efficiently repaired. We have examined the transcription and chromatin structure of these regions in growth-arrested cells to allow a more detailed comparison of transcription, nucleosome stability, and excision repair efficiency. Northern analysis, using strand-specific probes, indicates that four different transcripts are made from YRpTRURAP in addition to the URA3 mRNA in both growing and growth-arrested cells. The templates for these transcripts encompass all of the efficiently repaired regions outside of the URA3 gene. Nucleosome mapping indicates that the structure of the minichromosome in growth-arrested cells is indistinguishable from that of growing cells except for two nucleosomes in the region 500-800 base pairs 5' of the URA3 gene which become much less stable in growth-arrested cells. Comparison of the distribution of YRpTRURAP topoisomers in the two states, however, indicates that these nucleosomes are not lost from the majority of plasmid molecules. One of the four transcripts initiates in this region and increases by more than 5-fold in growth-arrested cells. Another transcript extends into a "slowly repaired" region which contains a very stable nucleosome. By determining the stability and relative amounts (at equilibrium) of the RNAs made from YRpTRURAP, transcription rates were determined and compared with the average PD repair rate for the different template regions. The results indicate that: 1) the rate of excision repair increases once a low, basal rate of transcription is achieved; 2) beyond this rate of transcription there is no simple correlation between the rates of transcription and PD repair; 3) nucleosome stability may "override" the coupling between transcription and repair if the transcription rate is low; and 4) at higher transcription rates, repair may be insensitive to nucleosome stability.
Base Sequence↗
Species specific protein--DNA interactions may determine the chromatin units of genes in S.cerevisiae and in S.pombe.
Yeast genes, such as URA3, are chromatin units characterized by positioned nucleosomes and flanking nuclease sensitive regions (NSRs). To investigate the structural determinants at the chromatin level in vivo, the URA3 gene was dissected into three parts (U5', Umid and U3'), and the chromatin structures of the individual parts were analysed after insertion into minichromosomes and after chromatin assembly in vivo in Saccharomyces cerevisiae. While nucleosome positions were altered on Umid, the 5'-end and the 3'-end of URA3 maintained their native structures (a positioned nucleosome and a NSR each) independent of the site or orientation of insertion. This suggests that the chromatin unit of the native URA3 gene is dominated by strong protein boundaries at the 5'- and 3'-ends. In an alternative approach, we investigated whether nucleosome positions or NSRs were maintained when the whole URA3 gene was placed on a shuttle vector and assembled into chromatin by Schizosaccharomyces pombe providing different proteins, but the same nucleosomal spacing. In a complementary exchange experiment, the ade6 gene of S.pombe was shuttled to S.cerevisiae. In spite of a general conservation of histone proteins and nucleosome core structures, neither nucleosome positions nor NSRs were maintained in the heterologous background. The results demonstrate that chromatin structures are species specific and that the structural boundaries of yeast genes may be dominated by strong species specific protein-DNA interactions.
Chromatin↗
Artificial nucleosome positioning sequences tested in yeast minichromosomes: a strong rotational setting is not sufficient to position nucleosomes in vivo.
DNA sequences that support bending around the histone octamer ('rotational setting') are considered to be a major determinant of nucleosome positions. TG5 is an artificial positioning sequence containing 100 bp of an (A/T)3NN(G/C)3NN motif repeated with a 10 bp period. It provides a strong rotational setting and is superior to natural sequences in nucleosome formation in vitro [Shrader, T.E. and Crothers, D.M. (1989) Proc. Natl. Acad. Sci. USA, 86, 7418-7422]. To investigate the contribution of the rotational setting to nucleosome positioning in vivo, TG sequences were inserted in a nucleosome, at the edge of a nucleosome and in a nuclease sensitive region of yeast minichromosomes and the chromatin structures were analysed. In none of the constructs were TG sequences folded in a positioned nucleosome, demonstrating that the rotational setting played a subordinate role in the rough positioning in vivo. The rotational setting might fine tune the positions. Positioned nucleosomes were found overlapping the ends of TG, indicating that a discontinuity of the 10 bp periodicity of (A/T)3 and (G/C)3 near the centre of a nucleosome might be favourable for positioning and serve as a translational signal.
Base Sequence↗
Structural changes in nucleosomes during transcription: strip, split or flip?
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DNA-Directed RNA Polymerases↗
Poly(dA).poly(dT) rich sequences are not sufficient to exclude nucleosome formation in a constitutive yeast promoter.
It was suggested that poly(dA).poly(dT) rich sequences in yeast Saccharomyces cerevisiae act as elements of constitutive promoters by exclusion of nucleosomes (Struhl, K. (1985). Proc. Natl. Acad. Sci. USA 82, 8419-8423). We have mapped the chromatin structure of the pet56-his3-ded1 region in minichromosomes and show that the poly(dA).poly(dT) sequences are located in nuclease sensitive regions. DNA fragments from the nuclease sensitive promoter region of DED1 were used for nucleosome reconstitution in vitro. We show that all sequences can form nucleosome cores and that the poly(dA).poly(dT) sequence can be incorporated in nucleosome cores. The results suggest that the nuclease sensitivity found in vivo is not established by poly(dA).poly(dT) mediated exclusion of nucleosomes.
Base Sequence↗
Site-specific DNA repair at the nucleosome level in a yeast minichromosome.
The rate of excision repair of UV-induced pyrimidine dimers (PDs) was measured at specific sites in each strand of a yeast minichromosome containing an active gene (URA3), a replication origin (ARS1), and positioned nucleosomes. All six PD sites analyzed in the transcribed URA3 strand were repaired more rapidly (greater than 5-fold on average) than any of the nine PD sites analyzed in the nontranscribed strand. Efficient repair also occurred in both strands of a disrupted TRP1 gene (ten PD sites), containing four unstable nucleosomes, and in a nucleosome gap at the 5' end of URA3 (two PD sites). Conversely, slow repair occurred in both strands immediately downstream of the URA3 gene (12 of 14 PD sites). This region contains the ARS1 consensus sequence, a nucleosome gap, and two stable nucleosomes. Thus, modulation of DNA repair occurs in a simple yeast minichromosome and correlates with gene expression, nucleosome stability, and (possibly) control of replication.
Base Sequence↗
DNA repair in a small yeast plasmid folded into chromatin.
The question of whether excision repair of yeast plasmids accurately reflects the repair of yeast genomic chromatin has yielded conflicting answers. These conflicts could have arisen from differences in the conformation of plasmid molecules used during these studies. We have examined excision repair of UV photoproducts in a small (2619 bp) autonomously replicating plasmid (YRp-TRURAP), known to be folded into chromatin with positioned nucleosomes in vivo, in the yeast Saccharomyces cerevisiae. A quantitative assay was used to measure the yield of cyclobutane pyrimidine dimers (PD) in plasmid DNA by measuring the fraction of Form I molecules resistant to T4 endonuclease V. After a UV dose of 100 J/m2, which yields 1.2 PD/plasmid in irradiated cells, radiation insensitive (wt) cells repair approximately 70% of the PD in TRURAP chromatin in 2 hr (a rate comparable to that of genomic chromatin). On the other hand, no measurable repair occurs in TRURAP chromatin in radiation sensitive cells (rad1) during the same time period. Thus, this small plasmid contains sufficient chromatin structure in vivo to reflect the incompetent repair of genomic chromatin seen in a rad mutant, while maintaining the competent repair level in wt cells.
Base Sequence↗
Electron microscopy of chromatin.
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Animals↗
Chromatin folding modulates nucleosome positioning in yeast minichromosomes.
Based on the chromatin structures of the yeast URA3 gene and the TRP1ARS1 circle, we have designed circular minichromosomes of different sizes that should each form a tight tetranucleosome. This structure was assumed to be stiff and bulky and therefore likely to be sensitive to packaging into a three-dimensional structure. The structures of the minichromosomes were determined using micrococcal nuclease. Only one of the minichromosomes showed a protected region of about 570 bp, compatible with the predicted tight tetranucleosome, while all other constructs showed alternative structures. A comparison of the structures revealed that neither histone-DNA interactions nor influences from flanking boundaries are sufficient determinants of nucleosome positions. The data strongly suggest that chromatin folding modulates the nucleosome arrangement along the DNA.
Chromatin↗
Topoisomerase I cleavage sites identified and mapped in the chromatin of Dictyostelium ribosomal RNA genes.
Sites of an endogenous activity that has the properties of a DNA topoisomerase I have been identified on the palindromic ribosomal RNA genes of the slime mould Dictyostelium discoideum. This was done in vitro, by treating isolated nuclei with sodium dodecyl sulphate, which denatures topoisomerase during its cycle of nicking, strand passing and resealing, and hence reveals the DNA cleavages. It was also done in vivo using the drug camptothecin, which is believed to stabilize the cleavable complex of topoisomerase I plus DNA, hence increasing the chances of cleavage when sodium dodecyl sulphate is subsequently added. The cleavages in vitro and in vivo were mapped by indirect end-labelling. Both treatments cause what appear to be strong double-stranded cleavages at 200 and 2200 base-pairs and at 17 X 10(3) base-pairs upstream from the rRNA transcription start. The cleavage at 200 base-pairs was analysed in greater detail using RNA hybridization probes specific for single DNA strands. The cleavage is in fact composed of three closely spaced nicks on each DNA strand. The DNA sequence at each of the nicks is strongly homologous across 15 base-pairs. Sodium dodecyl sulphate-induced cleavage by eukaryotic topoisomerase I is known to yield enzyme covalently attached to the 3' cut end of the DNA. We show that protein-linked DNA restriction fragments with their 3' ends at the cleavage sites are selectively retarded on denaturing gels, which provides strong evidence that the unusual cluster of cleavages is caused by a topoisomerase I. Additionally, the camptothecin results revealed cleavages not only at the specific upstream sites, but also across the transcribed region. Interestingly, the zone of camptothecin-assisted cleavage does not extend as far at the 3' end of the gene as the zone of endogenous nuclease sensitivity.
Base Sequence↗
Structure of the DNA gyrase-DNA complex as revealed by transient electric dichroism.
We have analyzed the structure of complexes between DNA gyrase and four defined DNA fragments by electric dichroism. Both the extrapolated dichroism and relaxation time of these complexes suggest that a single turn of DNA is wrapped around the enzyme with the entry and exit points located close together. The average angle between the DNA tails emerging from the particle is about 120 degrees. This structure is consistent with that seen by electron microscopy. Addition of ATP or the non-hydrolyzable ATP analog 5'-adenylyl-beta, gamma-imidodiphosphate results in a structural change of the complex, consistent with the DNA tails now being wrapped around the protein. The significance of these observations with respect to the mechanism of DNA supercoiling by DNA gyrase is discussed.
Autoradiography↗
Protein-DNA interactions and nuclease-sensitive regions determine nucleosome positions on yeast plasmid chromatin.
To study mechanisms of nucleosome positioning, small circular plasmids were constructed, assembled into chromatin in vivo in Saccharomyces cerevisiae, and their chromatin structures were analysed with respect to positions of nucleosomes and nuclease-sensitive regions. Plasmids used include insertions of the URA3 gene into the TRP1 gene of the TRP1ARS1 circular plasmid in the same (TRURAP) or opposite (TRARUP) orientation. The URA3 gene has six precisely positioned, stable nucleosomes flanked by nuclease-sensitive regions at the 5' and 3' ends of the gene. Three of these nucleosome positions do not depend on the flanking nuclease-sensitive regions, since they are formed at similar positions in a derivative plasmid (TUmidL) that contains the middle of the URA3 sequence but not the 5' and 3' ends. These positions are probably due to protein-DNA interactions. In both TRURAP and TRARUP, the positions of the nucleosomes on the TRP1 gene were, however, shifted compared with the positions on the parental TRP1ARS1 circle and TUmidL. These changes are interpreted to be due to changes in the positions of flanking nuclease-sensitive regions that might act as boundaries to position nucleosomes. Thus, two independent mechanisms for nucleosome positioning have been demonstrated in vivo. The ARS1 region contains the 3' end of the TRP1 gene and the putative origin of replication. Since in TRURAP and TRARUP the TRP1 gene is interrupted, but the ARS1 region remains nuclease sensitive, this non-nucleosomal conformation of the ARS1 region probably reflects a chromatin structure important for replication.
Base Sequence↗