Chromatin is the template of all processes involved in DNA metabolism in the eukaryotic cell. Accordingly, chromatin is a dynamic structure which changes in its composition and posttranslational modification, correlating with the functional state of a genomic locus (13
). It is important to understand how chromatin structure is established, maintained, and altered, thereby controlling the access to the genetic information, and to identify factors and molecular mechanisms involved in these processes.
An excellent example to study the correlation between transcription and chromatin structure is the ribosomal DNA (rDNA) locus in Saccharomyces cerevisiae
(hereafter called yeast). The rDNA is located on the right arm of chromosome XII and consists of 150 to 200 transcription units arranged head to tail in a tandem array (31
) (Fig. ). Each of these repeated units is composed of the RNA polymerase I (Pol I)-transcribed 35S rRNA gene (precursor for the 18S, 5.8S, and 25S rRNAs) and two intergenic spacers (IGS1 and IGS2). IGS1 contains the transcriptional enhancer (ENH) for 35S rRNA transcription and is separated from IGS2 by the 5S rRNA gene transcribed by RNA polymerase III (Pol III) (Fig. ). Interestingly, the 35S rRNA genes coexist in (at least) two different chromatin states in an actively dividing yeast cell (11
). Actively transcribed 35S rRNA genes are largely devoid of histone molecules and bound by the HMG box protein Hmo1, whereas transcriptionally inactive 35S rRNA genes are nucleosomal (26
FIG. 1. Schematic representation of the yeast rDNA locus. The position of the rDNA repeat cluster on chromosome XII with respect to the centromere (CEN) and telomeres (tel) is shown. Each rDNA repeat consists of the Pol I-transcribed 35S rRNA gene (precursor (more ...)
Efficient transcription initiation by Pol I requires the following four transcription factors, forming together with the RNA polymerase a preinitiation complex (PIC): upstream activating factor (UAF), core factor (CF), TATA-binding protein (TBP; yeast Spt15), and Rrn3 (31
). Another factor, Net1, a component of the regulator of nucleolar silencing and telophase exit (RENT) complex (see below), localizes at the Pol I promoter and stimulates Pol I transcription both in vitro
and in vivo
). Reb1, a ubiquitous DNA-binding protein factor, has two recognition sites in rDNA, within the ENH region and at the 5′ end of the Pol I promoter (27
) (Fig. ). Interestingly, both of the Reb1 recognition sites, but especially the promoter-proximal DNA element, contribute to efficient 35S rRNA gene transcription in vivo
UAF is a multiprotein complex binding to the upstream element (UE) of the Pol I promoter (Fig. ). The complex consists of the following six subunits: Rrn5, Rrn9, Rrn10, Uaf30, and the histones H3 and H4 (18
). Uaf30 was demonstrated to be important for UAF recruitment to the UE (15
), whereas the functions of the other factors (besides mediating specific protein-protein interactions [40
]) are still unknown. It is generally accepted that UAF nucleates PIC formation. However, it is under discussion whether UAF, TBP, and CF can independently form a stable complex at the Pol I promoter (19
) or if CF together with Rrn3-bound Pol I cycles on and off the promoter over the course of each initiation event (2
). In good agreement with in vitro
data, UAF plays an important role for Pol I transcription in vivo
, and in the absence of single UAF subunits, 35S rRNA production is severely impaired (19
Apart from its role in stimulating Pol I transcriptional activity, UAF plays an important role in inhibiting transcription of 35S rRNA genes by RNA polymerase II (Pol II). Deletion of any of the genes coding for the subunit Rrn5, Rrn9, or Rrn10 prevents Pol I transcription of 35S rRNA genes, which are then transcribed by Pol II from a cryptic promoter upstream from the RNA Pol I initiation site (43
). This polymerase switch (PSW) is accompanied by an increase in the copy number of rDNA repeats (32
). Deletion of the gene coding for Uaf30 leads to transcription of 35S rRNA genes by both Pol I and Pol II, without significant changes in rDNA repeat copy number, but can also result in a PSW phenotype under certain conditions (38
). The fact that a single factor controls promoter usage by different RNA polymerases is so far a unique feature of UAF, but the mechanism remains ill defined.
UAF is also important for silencing of Pol II reporter genes integrated into the rDNA locus (9
) and the Pol II-dependent production of noncoding RNAs (ncRNAs) within IGS1 and IGS2 (7
). Efficient silencing of Pol II transcription in rDNA further depends on Pol I transcription (7
) and on the histone deacetylase Sir2 together with Net1 and Cdc14 forming the RENT complex (4
). This has led to the model that UAF, perhaps together with other Pol I-associated factors, nucleates a special chromatin structure at the Pol I promoter, being repressive for 35S rRNA synthesis by Pol II and spreading to other rDNA regions by factors like RENT (9
). However, an in-depth molecular characterization of the UAF-dependent rDNA chromatin structure is still missing.
In this study, we investigated rDNA chromatin structure and composition upon deletion of components of the Pol I transcription machinery. We found that deletion of UAF results in a reorganization of Pol I promoter chromatin. In the absence of UAF, flanking regions of the promoter-proximal Reb1 binding site become accessible for binding of Pol II and III and associated transcription factors and may be sites of transcription initiation in these strains. Furthermore, the integrity of the UAF complex is required for the association of Sir2 with the rDNA locus, which can explain defective silencing of Pol II transcription upon deletion of UAF components. We demonstrate that alterations in chromatin composition extend throughout the 35S rRNA coding sequence. Interestingly, none of the above-described changes can be provoked by short-term inactivation of Pol I transcription. Taken together, our analyses shed light on how UAF organizes rDNA chromatin, thereby determining RNA polymerase specificity at the Pol I promoter and rDNA silencing of Pol II transcription.