Search

Glucose depletion derepresses the Saccharomyces cerevisiae ADH2 gene; this metabolic change is accompanied by chromatin structural modifications in the promoter region. We show that the ADR6/SWI1 gene is not necessary for derepression of the wild type chromosomal ADH2, whereas the transcription factor Adr1p, which regulates several S. cerevisiae functions, plays a major role in driving nucleosome reconfiguration and ADH2 expression. When we tested the effect of individual domains of the regulatory protein Adr1p on the chromatin structure of ADH2, a remodeling consisting of at least two steps was observed. Adr1p derivatives were analyzed in derepressing conditions, showing that the Adr1p DNA binding domain alone causes an alteration in chromatin organization in the absence of transcription. This alteration differs from the remodeling observed in the presence of the Adr1p activation domain when the promoter is transcriptionally active.

The chromatin organization of eukaryotic telomeres is essential for telomeric function and is currently receiving great attention. In yeast, the structural organization of telomeres involves a complex interplay of telomeric proteins that results in the formation of heterochromatin. This telomeric heterochromatin involves homotypic and heterotypic protein interactions that have been summarized in a general model. Recent analyses have focused on the study of the structural complexity at yeast telomeres to the level of specific nucleosomes and of the distribution of protein complexes in a natural telomeric region (LIII). In this report, we further analyze the structural complexity of LIII and the implication of this structure on telomeric silencing. It is shown that the establishment of repressive heterochromatin structures at LIII requires the recruitment of Sir3p through interaction with the N terminus of histone H4. The establishment of such structures does not require acetylation of any of four lysines located in the H4 N terminus (lysines 5, 8, 12, and 16).

We have defined the in vivo heterochromatin structure of the left telomere of Saccharomyces cerevisiae chromosome III (LIII). Analysis of heterochromatin of a single telomere was so far lacking, due to the difficulties intrinsic to the highly repetitive nature of telomeric sequences. In LIII, the terminal (C1-3A)n repetitive sequences are followed by a complete X element and by the single copy Ty5-1 retrotransposon. Both the telosome and the X element exhibit overall resistance to micrococcal nuclease digestion reflecting their tight chromatin structure organization. The X element contains protein complexes and irregularly distributed but well localized nucleosomes. In contrast, a regular array of phased nucleosomes is associated with the promoter region of Ty5-1 and with the more centromere-proximal sequences. The lack of a structural component of yeast telomeres, the SIR3 protein, does not alter the overall tight organization of the X element but causes a nucleosome rearrangement within the promoter region of Ty5-1 and releases Ty5-1 silencing. Thus, Sir3p links the modification of the heterochromatin structure with loss of transcriptional silencing.

The chromatin structure of the Saccharomyces cerevisiae ADH2 gene is modified during the switch from repressing (high glucose) to derepressing (low glucose) conditions of growth. Loss of protection toward micrococcal nuclease cleavage for the nucleosomes covering the TATA box and the RNA initiation sites (-1 and +1, respectively) is the major modification taking place and is strictly dependent on the presence of the transcriptional activator ADR1. To identify separate functions involved in the transition from a repressed to a transcribing promoter, we have analyzed the ADH2 chromatin organization in various genetic backgrounds. Deletion of the CCR4 gene coding for a general transcription factor impaired ADH2 expression without affecting chromatin remodeling. Growing yeast at 37 degrees C also resulted in chromatin remodeling at the ADH2 locus even under glucose repressing conditions. However, although this temperature-induced remodeling was dependent on the ADR1 protein, no ADH2 mRNA was observed. In addition, inactivating RNA polymerase II (and therefore, elongation) was found to have no effect on the ability to reconfigure nucleosomes. Taken together, these data indicate that chromatin remodeling by itself is insufficient to induce transcription at the ADH2 promoter.

A 569-base pair fragment encompassing the upstream regulatory region, the RNA initiation sites, and the initial part of the coding region of the Saccharomyces cerevisiae alcohol dehydrogenase II gene has been analyzed for the presence of sites which undergo conformational modification under torsional stress. Fine mapping of P1 and S1 endonuclease-sensitive sites was obtained on single topoisomers produced by in vitro ligation. It was shown that the upstream activator sequence, the TATA sequence, a region directly upstream to the RNA initiation sites, and several positions in the first segment of the transcribed region change conformation as a function of the applied torsional stress in a precisely coordinate fashion. The superhelical density optima for this coordinate modifications have been determined. Analysis of the conformational changes of the promoter sequence in several naturally occurring (Young, E. T., Williamson, V. M., Taguchi, A., Smith, M., Sledziewski, L., Russel, D., Osterman, J., Denis, C., Cox, D., and Beier, D., (1982) in Genetic Engineering of Microorganisms for Chemicals (Hollander, A., De Moss, R. D., Kaplan, S., Konisky, J., Savage, D., and Wolle, R. S., eds) pp. 335-361, Plenum Publishing Corp., New York) up-promoter constitutive mutants was performed. This analysis has shown that the conformation of functionally relevant sites changes as a function of sequence mutations that have taken place elsewhere; this shows that the conformational behavior of the whole promoter region is linked and suggests transmission in cis of topological effects in RNA polymerase II promoters.

We present an analysis of the influence of DNA superhelicity on the topology of chimeric plasmids. A correlation is found between topological variations and in vivo/in vitro functions. Data refer to the topological transitions observed in function of variations of the superhelical density in the three closed DNA domains that represent the three extreme examples, from the point of view of the topological organization, among many chimeric systems analyzed. The plasmids studied are ADR2-BS-pBR322 (the vector pBR322 + the yeast gene ADR2), ADR3-5c-yRp7 (containing yeast TRP1, a constitutive mutant of yeast ADR2, a fragment of 5 kilobases of unanalyzed chromosomal DNA and the vector pBR322), and p31 (pBR322 + a yeast DNA fragment encompassing the right moiety of the Ty1 element and its in vivo promoter). In the model systems analyzed, the topological transitions observed in the eukaryotic sequences (relevant in one case for activation of selective in vitro transcription (Ty), in another (ADR2) for the in vivo expression) take place in the range of superhelical densities predicted on theoretical ground (Vologodskii, A. V., Lukashin, A. V. Anshelevich, V. V. & Frank-Kamenetskii, M. D. (1979) Nucleic Acids Res. 6, 967-982).

Activation of in vitro transcription of otherwise inert DNA sequences by purified yeast RNA polymerase II has been observed following the introduction in closed DNA domains of fragments of various origin. This enhancer-like effect on the in vitro transcriptional capacity is only detected in negatively supercoiled DNA domains and is characterized for each chimaeric plasmid by the superhelical density (- sigma) at which a sharp transition toward activation takes place. We have analyzed the topological state (as defined by localization and evaluation of the relative occurrence of secondary structures sensitive to S1 endonuclease) of the activated closed domains as a function of the conditions that determine the transcriptional enhancer effect, i.e. superhelical density, size, and nature of the components of the domains. We observe that variations in transcriptional capacity coincide with a defined pattern of secondary structures. These observations support a cause-effect relation between topology and regulation of transcription.

Different conformations of the circular DNA domains containing the intergenic region of the Saccharomyces cerevisiae GAL1-GAL10 divergent genes (914 base pairs) were modified by in vitro ligation in various conditions. The effect of increasing torsional stress on the conformation of the composing elements was determined by analysis of the sensitivity to the single strand-specific S1 endonuclease and it was observed that the sites of conformational alterations correspond to the positions relevant for promoter function (upstream activator sequence, TATA sequence, and RNA initiation site).

In vivo nucleotide-level mapping of nucleosomes in the promoter of the methanol oxidase (MOX) gene in the yeast Hansenula polymorpha is reported. The 4 nucleosomes analyzed are organized in families; they localize in alternative positions along a unique rotational phase, and the linker regions can be occupied by alternative nucleosomes. This organization underscores a substantial freedom of choice by histone octamers when nucleating on a promoter region.