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Mapping of MNase cleavage sites on reconstituted chromatin templates. Chromatin assembled on plasmid pMrWT (lanes 1 and 2) and chromatin incubated with 10 ng TTF-I after completion of chromatin assembly (lanes 3 and 4), was digested with 10 U MNase for 10 and 30 s. In the reactions represented in lanes 5 and 6, the reconstituted chromatin was treated with apyrase before TTF-I addition. After cleavage with NdeI, electrophoresis and blotting, hypersensitive sites were visualized by hybridization to a 207 bp EcoRI–NdeI fragment derived from pUC9. Predominant nucleosome positions at the rDNA promoter region are indicated by open circles; the transcription start site is indicated by a filled arrow; the TTF-I binding site is indicated by an open arrow.
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We have analyzed the chromatin structure of mouse ribosomal RNA genes (rDNA) by partial digestion of genomic DNA with micrococcal
nuclease (MNase), DNase I and identified hypersensitive sites by indirect end-labeling. This analysis has revealed defined
regions of nuclease hypersensitivity in the intergenic spacer which in turn coincide with regulat...
Context in source publication
Context 1
... chromatin reconstitution was performed on pMrWT and recombinant TTF-I was added after completion of chromatin assembly. The MNase digestion pattern of the in vitro reconstituted chromatin is shown in Figure 5. The cleavage pattern of chromatin assembled in the absence of TTF-I closely resembles that of naked DNA (data not shown) indicating that the nucleosomes are randomly distributed on the rDNA plasmid (lanes 1 and 2). ...
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We have used cell-free DNA replication to study the relationship between DNA replication and chromatin assembly. As others have reported, we find that DNA replication facilitates nucleosome assembly. We show here that replication-dependent nucleosome assembly occurs in at least two steps. The first step requires replicating DNA; the second step occ...
Retinoic acid (RA) stimulates transcription from the retinoic acid receptor beta2 (RARbeta2) promoter in mammalian embryonal cells. Evidence by in vivo deoxyribonuclease I (DNase I) hypersensitivity assay indicates that RA treatment of these cells results in an alteration of chromatin structure in and near the promoter. To study the role of chromat...
Citations
... Structural modelling of the rDNA promoter region from yeast and other organisms has predicted a bend and sequence-dependent flexibility in the upstream region of the DNA, important for promoter recognition 9,15 . Randomization of the promoter sequence also showed that the region from base pair (bp) −30 to −1 is critical for in vitro transcription 9 consistent with the observation that CF recognizes the promoter from bp −27 to −16 (ref. ...
RNA polymerase I (Pol I) assembles with core factor (CF) and Rrn3 on the rDNA core promoter for transcription initiation. Here, we report cryo-EM structures of closed, intermediate and open Pol I initiation complexes from 2.7 to 3.7 Å resolution to visualize Pol I promoter melting and to structurally and biochemically characterize the recognition mechanism of Pol I promoter DNA. In the closed complex, double-stranded DNA runs outside the DNA-binding cleft. Rotation of CF and upstream DNA with respect to Pol I and Rrn3 results in the spontaneous loading and opening of the promoter followed by cleft closure and positioning of the Pol I A49 tandem winged helix domain (tWH) onto DNA. Conformational rearrangement of A49 tWH leads to a clash with Rrn3 to initiate complex disassembly and promoter escape. Comprehensive insight into the Pol I transcription initiation cycle allows comparisons with promoter opening by Pol II and Pol III.
... This interaction mediates at least four different functions namely termination of DNA replication (29)(30)(31), activation of transcription (8,32), termination of transcription, and a long-range chromosome-to-chromosome interaction called "chromosome kissing" (31). The mammalian ortholog TTF-I also performs similar functions (26,27,33,34). ...
Significance
Transcription termination of rRNA genes by RNA polymerase I (pol I) in fission yeast requires the binding of the Reb1 protein to a terminator site (Ter). Termination is physiologically necessary because its elimination can cause replication–transcription collision and induction of genome instability. Furthermore, without termination, pol I can become unproductively sequestered on the DNA templates. We have determined the crystal structure of fission yeast terminator protein Reb1-Ter complex revealing its functional architecture. Structure-guided functional analysis revealed that it is not just tight binding of the protein to Ter but protein–protein interactions with the Rpa12 subunit of RNA polymerase I that causes transcriptional arrest.
... A common feature of ribosomal gene promoters is that they lack sequence homology but retain structural similarity and contain intrinsically distorted regions [24]. The relative DNA curvature of the mouse rDNA promoter was calculated with the Bolshoy algorithm using the 'bandit' program ( Figure 3B, [25,26]). The mouse rRNA gene promoter contains a region of high local DNA curvature ( [25]; at about position 2110) that is specifically bound by Tip5 ( Figure 3C). ...
... The relative DNA curvature of the mouse rDNA promoter was calculated with the Bolshoy algorithm using the 'bandit' program ( Figure 3B, [25,26]). The mouse rRNA gene promoter contains a region of high local DNA curvature ( [25]; at about position 2110) that is specifically bound by Tip5 ( Figure 3C). This result agrees with the results of the previous experiment, which demonstrated the preferential binding of Tip5 to cruciform DNA ( Figure 1E). ...
Active and repressed ribosomal RNA (rRNA) genes are characterised by specific epigenetic marks and differentially positioned nucleosomes at their promoters. Repression of the rRNA genes requires a non-coding RNA (pRNA) and the presence of the nucleolar remodeling complex (NoRC). ATP-dependent chromatin remodeling enzymes are essential regulators of DNA-dependent processes, and this regulation occurs via the modulation of DNA accessibility in chromatin. We have studied the targeting of NoRC to the rRNA gene promoter; its mechanism of nucleosome positioning, in which a nucleosome is placed over the transcription initiation site; and the functional role of the pRNA. We demonstrate that NoRC is capable of recognising and binding to the nucleosomal rRNA gene promoter on its own and binds with higher affinity the nucleosomes positioned at non-repressive positions. NoRC recognises the promoter nucleosome within a chromatin array and positions the nucleosomes, as observed in vivo. NoRC uses the release mechanism of positioning, which is characterised by a reduced affinity for the remodeled substrate. The pRNA specifically binds to NoRC and regulates the enzyme by switching off its ATPase activity. Given the known role of pRNA in tethering NoRC to the rDNA, we propose that pRNA is a key factor that links the chromatin modification activity and scaffolding function of NoRC.
... Our group has previously determined the transcription start point (TSP) of the rRNA transcription unit [6], but the promoter and the transcription termination elements have not been characterised. Analysis of the intergenic sequence of the T. vaginalis ribosomal genes has revealed a lack of the repetitive or palindromic sequences (8-20 bp in length) that are known to regulate levels of transcription in other species [7][8][9][10]. Similarly, Sal boxes, which are known transcription terminating sequences in higher eukaryotes, are also not present [11]. ...
Trichomonas vaginalis is a parasitic protozoan of both medical and biological relevance. Transcriptional studies in this organism have focused mainly on type II pol promoters, whereas the elements necessary for transcription by polI or polIII have not been investigated. Here, with the aid of a transient transcription system, we characterised the rDNA intergenic region, defining both the promoter and the terminator sequences required for transcription. We defined the promoter as a compact region of approximately 180 bp. We also identified a potential upstream control element (UCE) that was located 80 bp upstream of the transcription start point (TSP). A transcription termination element was identified within a 34 bp region that was located immediately downstream of the 28S coding sequence. The function of this element depends upon polarity and the presence of both a stretch of uridine residues (U's) and a hairpin structure in the transcript. Our observations provide a strong basis for the study of DNA recognition by the polI transcriptional machinery in this early divergent organism.
... In contrast, ADP-ribose synthesis was not detected at the promoters of brca1 and -actin (Fig. 7C). In addition, despite the presence of nucleosomes at the rDNA promoter (33,65), ADP-ribose synthesis was not detected at this promoter. As expected, the addition of PJ34 abrogated ADP-ribose synthesis at the il-1, mip-2, and csf2 promoters (Fig. 7C). ...
The packaging of DNA into nucleosomes imposes obstacles on gene transcription, and histone-modifying and nucleosome-remodeling
complexes work in concert to alleviate these obstacles so as to facilitate transcription. Emerging evidence shows that chromatin-associated
poly(ADP-ribose) polymerase 1 (PARP-1) and its enzymatic activity facilitate inflammatory gene transcription and modulate
the inflammatory response in animal models. However, the molecular mechanisms by which PARP-1 enzymatic activity facilitates
transcription are not well understood. Here we show that through an intracellular signaling pathway, lipopolysaccharide (LPS)
stimulation induces PARP-1 enzymatic activity and the ADP-ribosylation of histones at transcriptionally active and accessible
chromatin regions in macrophages. In vitro DNase I footprinting and restriction endonuclease accessibility assays reveal that histone ADP-ribosylation directly destabilizes
histone-DNA interactions in the nucleosome and increases the site accessibility of the nucleosomal DNA to nucleases. Consistent
with this, LPS stimulation-induced ADP-ribosylation at the nucleosome-occupied promoters of il-1β, mip-2, and csf2 facilitates NF-κB recruitment and the transcription of these genes in macrophages. Therefore, our data suggest that PARP-1
enzymatic activity facilitates gene transcription through increasing promoter accessibility by histone ADP-ribosylation.
... This finding corresponds to positions 46/56 to 196/206 (N1) on the 248-bp rDNA fragment studied in SI Fig. 7A. Previous studies established that the rDNA promoter of a variety of organisms exhibits a conserved sequence-dependent structure (32,33). An analysis of this sequence for specific features revealed a strong correlation between ACF-dependent nucleosome positioning and the presence of an intrinsically curved DNA region (Fig. 3D). ...
Chromatin-remodeling complexes can translocate nucleosomes along the DNA in an ATP-coupled reaction. This process is an important regulator of all DNA-dependent processes because it determines whether certain DNA sequences are found in regions between nucleosomes with increased accessibility for other factors or wrapped around the histone octamer complex. In a comparison of seven different chromatin-remodeling machines (ACF, ISWI, Snf2H, Chd1, Mi-2, Brg1, and NURF), it is demonstrated that these complexes can read out DNA sequence features to establish specific nucleosome-positioning patterns. For one of the remodelers, ACF, we identified a 40-bp DNA sequence element that directs nucleosome positioning. Furthermore, we show that nucleosome positioning by the remodelers ACF and Chd1 is determined by a reduced affinity to the end product of the translocation reaction. The results suggest that the linkage of differential remodeling activities with the intrinsic binding preferences of nucleosomes can result in establishing distinct chromatin structures that depend on the DNA sequence and define the DNA accessibility for other protein factors.
• ACF
• nucleosome remodeling
• nucleosome positioning
... In Xenopus, this T3 terminator (46), similar to the T 0 sequences in mouse (50), have been shown to augment transcription from the adjacent rDNA promoter (30,(51)(52)(53). The interaction of TTF-1 (transcription terminator factor) with terminator elements (54 -56) in a chromatin-and ATP-dependent fashion (57)(58)(59), activates RNA pol I transcription by a mechanism that remains to be determined. This T3 terminator is present in the minigene construct (pol I mini-CAT) that we used in this study. ...
Nucleolin is one of the most abundant non-ribosomal proteins of the nucleolus. Several studies in vitro have shown that nucleolin is involved in several steps of ribosome biogenesis, including the regulation of rDNA transcription, rRNA processing, and ribosome assembly. However, the different steps of ribosome biogenesis are highly coordinated, and therefore it is not clear to what extent nucleolin is involved in each of these steps. It has been proposed that the interaction of nucleolin with the rDNA sequence and with nascent pre-rRNA leads to the blocking of RNA polymerase I (RNA pol I) transcription. To test this model and to get molecular insights into the role of nucleolin in RNA pol I transcription, we studied the function of nucleolin in Xenopus oocytes. We show that injection of a 2-4-fold excess of Xenopus or hamster nucleolin in stage VI Xenopus oocytes reduces the accumulation of 40 S pre-rRNA 3-fold, whereas transcription by RNA polymerase II and III is not affected. Direct analysis of rDNA transcription units by electron microscopy reveals that the number of polymerase complexes/rDNA unit is drastically reduced in the presence of increased amounts of nucleolin and corresponds to the level of reduction of 40 S pre-rRNA. Transcription from DNA templates containing various combinations of RNA polymerase I or II promoters in fusion with rDNA or CAT sequences was analyzed in the presence of elevated amounts of nucleolin. It was shown that nucleolin leads to transcription repression from a minimal polymerase I promoter, independently of the nature of the RNA sequence that is transcribed. Therefore, we propose that nucleolin affects RNA pol I transcription by acting directly on the transcription machinery or on the rDNA promoter sequences and not, as previously thought, through interaction with the nascent pre-rRNA.
... Sequence-dependent structure variations of DNA, in particular DNA curvature, play an important role in the organization and function of the genome. It is known, for instance, that DNA curves can organize the global structure of superhelical DNA (Laundon and Grith 1988;Pfannschmidt and Langowski 1998;Tsen and Levene 1997), and that curved segments participate in transcriptional regulation (Bracco et al. 1989;Griess et al. 1993;Kim et al. 1995;LaÈ ngst et al. 1997;Lavigne et al. 1992;Ohyama 1996;Perez-Martin et al. 1994;Rojo and Salas 1991). The possibility of DNA curvature was ®rst proposed byTrifonov and Sussman (1980), who pointed out that local kinks in phase with the helical repeat would cause the DNA helix axis to follow a globally bent path. ...
The rotational dynamics of short DNA fragments with or without intrinsic curvature were studied using time-resolved phase fluorimetry of intercalated ethidium with detection of the anisotropy. Parameters determined were the spinning diffusion coefficient of the DNA fragments about the long axis and the zero-time ethidium fluorescence anisotropy. We find a significant decrease in the spinning diffusion coefficient for all curved fragments compared to the straight controls. This decrease is likewise evident in rotational diffusion coefficients computed from DNA structures obtained by a curvature prediction program for these sequences. Using a hinged-cylinder model, we can identify the change in rotational diffusion coefficient with a permanent bend of 13-16 degrees per helix turn for the sequences studied. Moreover, for some of the curved fragments an increased flexibility has to be assumed in addition to the permanent bend in order to explain the data.
... of rDNA transcription. In active interphase cells, the transcription termination factor (TTF-1) (for a review, see Reeder and Lang, 1994) that binds terminator elements present both upstream and downstream of rDNAs, seems to play a crucial role in the global structural organization of the rDNA transcription units (Sander and Grummt, 1997) as well as in the position of the nucleosome close to the transcription start site (Längst et al., 1998(Längst et al., , 1997b. In addition, in agreement with the in vivo effect of the promoter-proximal terminator element (Mitchelson and Moss, 1987;Moss et al., 1992), TTF-1 was reported to be an activator of RNA pol I transcription (Längst et al., 1997a). ...
... Moreover, as for TTF-1, Reb1p performs other functions in the cell in addition to its role in RNA pol I termination. In particular, Reb1p can rearrange nucleosomes (Fedor et al., 1988) and this activity could remodel chromatin over the yeast promoter in a manner similar to the way TTF-1 appears to act at the level of the mouse promoter (Längst et al., 1997b). ...
The transcription termination factor TTF-1 exerts two functions in ribosomal gene (rDNA) transcription: facilitating initiation and mediating termination of transcription. Using HeLa cells, we show that TTF-1 protein is colocalized with the active transcription machinery in the nucleolus and also with the inactive machinery present in certain mitotic nucleolar organizer regions (NORs) when rDNA transcription is repressed. We also show that TTF-1 is specifically phosphorylated during mitosis in a manner dependent on the cdc2-cyclin B kinase pathway and on an okadaic acid-sensitive phosphatase. Interestingly, the mitotically phosphorylated form of TTF-1 appearing at the G(2)/M transition phase was more easily solubilized than was the interphase form. This indicates that the chromatin-binding affinity of TTF-1 appears to be different in mitotic chromosomes compared to the interphase nucleolus. Correlated with this, the other DNA-binding factor, UBF, which interferes with chromatin conformation in the rDNA promoter, was more strongly bound to rDNA during mitosis than at interphase. The reorganization of the mitotic rDNA promoter might be induced by phosphorylation of certain components of the rDNA transcription machinery and participate in silencing of rDNA during mitosis.
... trinucleotide vs. dinucleotide representation, electrophoresis vs. nucleosome data, etc.) and not to the models themselves. Considering the significance of these structural features, it is worth to mention that bendability/curvature characteristics are conserved in evolutionarily[31,35]and functionally related sequences[37], and they correlate well with regulatory sites known from experiment[38]. So one can predict that these methods will have a continued role in locating functional sites in genomes and will hopefully contribute to our understanding how the putative conformational signals may operate at the genomic level. Refinements of rod models, especially the incorporation of tetranucleotide-based description will probably increase the predictive accuracy and increase the scope of applications. ...
Bending is a local conformational micropolymorphism of DNA in which the original B-DNA structure is only distorted but not extensively modified. Bending can be predicted by simple static geometry models as well as by a recently developed elastic model that incorporate sequence dependent anisotropic bendability (SDAB). The SDAB model qualitatively explains phenomena including affinity of protein binding, kinking, as well as sequence-dependent vibrational properties of DNA. The vibrational properties of DNA segments can be studied by finite element analysis of a model subjected to an initial bending moment. The frequency spectrum is obtained by applying Fourier analysis to the displacement values in the time domain. This analysis shows that the spectrum of the bending vibrations quite sensitively depends on the sequence, for example the spectrum of a curved sequence is characteristically different from the spectrum of straight sequence motifs of identical basepair composition. Curvature distributions are genome-specific, and pronounced differences are found between protein-coding and regulatory regions, respectively, that is, sites of extreme curvature and/or bendability are less frequent in protein-coding regions. A WWW server is set up for the prediction of curvature and generation of 3D models from DNA sequences (http://www.icgeb.trieste.it/dna).
