Reversible changes in nucleosome structure and histone H3 accessibility in transcriptionally active and inactive states of rDNA chromatin

Prior, Christopher P.; Cantor, Charles R.; Johnson, Edward M.; Littau, Virginia C.; Allfrey, Vincent G.

doi:10.1016/0092-8674(83)90561-5

Cited by 238 publications

(101 citation statements)

References 41 publications

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“…This idea is consistent with the finding that UBF homodimers bend rDNA into 140-bp loops (11)(12)(13)(14). In addition, studies of NORs in Physarum polycephalum demonstrated that transcriptionally active NOR chromosome regions have a unique, unfolded chromatin architecture (41). A logical speculation based on these studies was that binding of UBF throughout the NORs could induce chromatin remodeling to assist their conversion to a transcriptionally active state.…”

Section: Discussionsupporting

confidence: 73%

Upstream binding factor association induces large-scale chromatin decondensation

Chen

Belmont

Huang

2004

Proc. Natl. Acad. Sci. U.S.A.

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The function of upstream binding factor (UBF), an essential component of the RNA polymerase (pol) I preinitiation complex, is unclear. Recently, UBF was found distributed throughout ribosomal gene repeats rather than being restricted to promoter regions. This observation has led to the speculation that one role of UBF binding may be to induce chromatin remodeling. To directly evaluate the impact of UBF on chromatin structure, we used an in vivo assay in which UBF is targeted via a lac repressor fusion protein to a heterochromatic, amplified chromosome region containing lac operator repeats. We show that the association of UBF with this locus induces large-scale chromatin decondensation. This process does not appear to involve common remodeling complexes, including SWI͞SNF and histone acetyltransferases, and is independent of histone H3 lysine 9 acetylation. However, UBF recruits the pol I-specific, TATA box-binding protein containing complex SL1 and pol I subunits. Our results suggest a working hypothesis in which the dynamic association of UBF with ribosomal DNA clusters recruits the pol I transcription machinery and maintains these loci in a transcriptionally competent configuration. These studies also provide an in vivo model simulating ribosomal DNA transactivation outside the nucleolus, allowing temporal and spatial analyses of chromatin remodeling and assembly of the pol I transcription machinery. R ibosomal RNA (rRNA) is encoded by tandem arrays of rDNA genes that are organized in human cells into nucleolar organizing regions (NORs) located on five chromosome pairs. A specific set of transcription factors is dedicated to transcription of rDNA into pre-rRNA that is subsequently processed into 28S, 18S, and 5.8S rRNAs. The rRNAs are packaged with ribosomal proteins to form the large and small subunits of ribosomes (1). Transcription of rDNA is highly specific and extensively regulated, involving a large number of proteins (1-3). Studies based mostly on in vitro assays suggest that transcriptional activation of rDNA involves association of the preinitiation complex with the promoter region followed by recruitment of other factors and RNA polymerase (pol) I subunits. The preinitiation complex has been shown to contain upstream binding factor (UBF) (4) and the TATA box-binding protein (TBP) containing complex SL1 (5).UBF is highly conserved in vertebrate cells from Xenopus to human. It contains five high mobility group boxes, an N terminus necessary for nuclear translocation, and a highly acidic C terminus essential for nucleolar localization (4, 6, 7). Although UBF is involved in pol I transcriptional activation, the mechanism by which it acts remains unclear. The consensus derived mostly from in vitro and some in vivo correlative studies places UBF at the early steps of rDNA transactivation (1, 2, 8). Analyses using in vitro transcription assays indicate that UBF initially binds DNA and is necessary for SL1 binding to form the preinitiation complex (9). Recently, UBF was discovered to literally coat DNA thro...

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Section: Discussionsupporting

confidence: 73%

Upstream binding factor association induces large-scale chromatin decondensation

Chen

Belmont

Huang

2004

Proc. Natl. Acad. Sci. U.S.A.

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“…The separation of active from inactive chromatin depends on the fact that nucleosomes along active genes unfold during transcription to reveal the previously shielded cysteinyl-SH groups of histone H3 molecules at the center of the nucleosome core (26). Consequently, a mixture of active and inactive nucleosomes can be separated by entrapping the SH-reactive nucleosomes on a mercurated agarose support.…”

Section: Resultsmentioning

confidence: 99%

Isolation of active genes containing CAG repeats by DNA strand invasion by a peptide nucleic acid.

Boffa

Carpaneto

Allfrey

1995

Proc. Natl. Acad. Sci. U.S.A.

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An amplification of tandem CAG trinucleotide sequences in DNA due to errors in DNA replication is involved in at least four hereditary neurodegenerative diseases. The CAG triplet repeats when translated into protein give rise to tracts of glutamine residues, which are a prominent feature of many transcription factors, including the TATA-binding protein of transcription factor TFIID. We have used a biotin-labeled, complementary peptide nucleic acid (PNA) to invade the CAG repeats in intact chromatin and then employed a method for the selective isolation of transcriptionally active chromatin restriction fragments containing the PNA-DNA hybrids. The PNA-containing chromatin fragments were captured on streptavidin-agarose magnetic beads and shown to contain all the CAGPNA hybrids of the active chromatin fraction. DNA hybridization experiments using a DNA probe specific for unique sequences downstream of the CAG-tandem repeats confirmed that the PNA-DNA hybrids contained the transcribed gene for the TATA-binding protein.In contrast, no hybridization signal was detected with a DNA probe specific for the c-myc protooncogene, which is amplified and transcriptionally active in COLO 320DM cells but lacks CAG tandem repeats.Peptide nucleic acids (PNAs) are structural homologues of DNA and RNA in which the entire phosphate-sugar backbone has been replaced by a flexible peptide backbone consisting of 2-aminoethyl glycine units linked to the purine and pyrimidine bases (1-3). The absence of phosphate groups in the PNA molecule facilitates its invasion of negatively charged DNA duplexes containing complementary base sequences (4-6). When PNA hybridizes to a targeted DNA sequence, one strand of DNA may be rapidly displaced to form a D-loop (1, 5, 6), while the PNA binds to its complementary DNA sequence by WatsonCrick base pairing (7). PNAs show greater discrimination and form more stable associations with DNA than the corresponding DNADNA duplexes (7). PNA invasion of DNA strands to form stable PNADNA hybrids has profound implications for both positive and negative control of gene activity. For example, it has been shown that DNA loops displaced as a consequence of PNA binding act as artificial transcription promoters (8), but PNA binding to the transcribed strand of simian virus 40 DNA blocks transcript elongation beyond the site of PNA DNA duplex formation (9).Here, we explore the potential of a biotinylated PNA probe to invade DNA triplet repeat sequences in the chromatin of transcriptionally active genes. By using a PNA specific for multiple CAG repeats, together with a method for the separation of transcriptionally active chromatin restriction fragments from inactive chromatin restriction fragments (10), we show that the PNA probe can hybridize effectively to CAG triplet repeats in intact chromatin and that transcriptionally active chromatin fragments containing the PNA-DNA hybrids can be captured quantitatively on streptavidin-coated magnetic beads.An amplification of CAG triplet repeats occurs in at least four i...

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“…"Split" nucleosomes generated this way were supposed to have a role in transcription. In fact, it is evident from a number of studies that activation of chromatin is accompanied by exposure of the normally buried cysteines of H3 (Prior et al, 1983;Cocco et al, 1986;Allegra et al, 1987;Chen & Allfrey, 1987;Johnson et al, 1987). Because changes leading to the opening of nucleosome structure are thought to be involved in activation of chromatin, a nucleosome carrying a fluorescence probe at its H3 molecules can also be considered as having a conformation close to that of the nucleosome of active chromatin.…”

Section: Discussionmentioning

confidence: 99%

Dynamics of interaction of RNA polymerase II with nucleosomes. II. During read‐through and elongation

Bhargava

1993

Protein Science

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The sulfhydryl-specific fluorescence probe 1,SIAEDANS (5-(2-((iodoacetyl)amino)ethyl)amino-naphthalene-1-sulfonic acid) was attached to the single cysteine of H3, and reconstituted fluorescent mononucleosomes were used as the template for in vitro transcription by the yeast RNA polymerase I1 (pol 11). DNase 1 digestion analysis revealed that transcription of nucleosomes by pol I1 resulted in an overall loosening of the structure. Monitoring the transcription event by steady-state fluorescence analysis showed that nucleosomes only partially open during transcription. This opening is transient in nature, and nucleosomes close back as soon as the pol I1 falls off the template. Thus, using the technique of fluorescence spectroscopy, partial opening of nucleosome structure could be differentiated from complete dissociation into free DNA and histone octamer, a distinction that may not be possible by techniques like gel electrophoresis. Time-resolved fluorescence emission spectroscopy suggested that during read-through of the template by the pol 11, histone octamers do not fall off the DNA. Only minor conformational changes within the histone octamer take place to accommodate the transcribing polymerase.

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Reversible changes in nucleosome structure and histone H3 accessibility in transcriptionally active and inactive states of rDNA chromatin

Cited by 238 publications

References 41 publications

Upstream binding factor association induces large-scale chromatin decondensation

Upstream binding factor association induces large-scale chromatin decondensation

Isolation of active genes containing CAG repeats by DNA strand invasion by a peptide nucleic acid.

Dynamics of interaction of RNA polymerase II with nucleosomes. II. During read‐through and elongation

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