Chapter 12 Nucleosomes of Transcriptionally Active Chromatin: Isolation of Template-Active Nucleosomes by Affinity Chromatography

Allfrey, Vincent G.; Chen, Thelma A.

doi:10.1016/s0091-679x(08)60578-6

Cited by 20 publications

(20 citation statements)

References 55 publications

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“…Consequently, a mixture of active and inactive nucleosomes can be separated by entrapping the SH-reactive nucleosomes on a mercurated agarose support. The mercury-bound nucleosomes (and larger chromatin fragments containing the SH-reactive nucleosomes) are recovered by displacement with DTT (27,28). When mercury column chromatography was used to detect changes in nucleosome structure during oncogene activation, it was revealed that nucleosomes along an activated c-fos gene unfolded within minutes and reverted to the compact nonmercury-binding form within 15 min after transcription was suppressed (29)(30)(31).…”

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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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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“…In all experiments, to ensure a minimum amount of background replication, the cells were grown to confluence and treated with 2 mM hydroxyurea 45 min prior to UV damage. To allow comparison of data between experiments and between samples within one experiment, the "double-label" procedure described in Smerdon et al (17) Isolation of Nuclei and Chromatin Preparation-Nuclei were isolated as described (24) with the following modifications. Harvested cells were pelleted and resuspended in a buffer containing 10 mM Tris, pH 8.0, 1.0 mM CaCl 2 , 8.6% sucrose, and 5.0 mM sodium butyrate (to inhibit histone deacetylation).…”

Section: Candmentioning

confidence: 99%

“…The thiol-reactive chromatin released in fraction 3 represents only 1.8 Ϯ 0.4% of the soluble chromatin, or about 0.4% of total chromatin (see below). In the transcription studies on human 3T3 cells, Allfrey and co-workers (24,29) found that ϳ10% of the soluble chromatin was recovered in fraction 2 and ϳ7% in fraction 3. These differences from our results may reflect differences in solubility of the fragment sizes produced.…”

Section: Organomercurial Fractionation Of Normal Human Chromatin-normmentioning

confidence: 99%

“…An Actively Transcribing Gene Is Preferentially Associated with the Thiol-reactive Fraction-Allfrey and co-workers have demonstrated that transcriptionally active genes are preferentially associated with the thiol-bound fraction (24). As a control for our experiments, the mercury bead fractions were tested for the presence of DHFR, which is constitutively expressed in the confluent human cells used in our experiments (35,36).…”

Section: Fig 1 Schematic Diagram Of Chromatin Fractionation Scheme mentioning

confidence: 99%

“…see Ref. 24) employed organomercurial affinity chromatography as a means to bind "open" nucleosomes at exposed thiols of disrupted (or unfolded) H3 histones to capture actively transcribing chromatin. Nucleosomes from a number of different species, including slime mold, yeast, mice, and humans, were fractionated with this method, and in each case active genes were preferentially bound to the organomercurial affinity matrix.…”

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Nucleosome Unfolding during DNA Repair in Normal and Xeroderma Pigmentosum (Group C) Human Cells

Baxter¹,

Smerdon

1998

Journal of Biological Chemistry

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The fate of nucleosomes during nucleotide excision repair is unclear. We have used organomercurial chromatography to capture accessible thiol groups of proteins at (or near) nascent repair sites in normal and xeroderma pigmentosum (group C) human cells. The reactive groups include cysteine 110 of histone H3, which is exposed in unfolded nucleosomes. Immediately after UV irradiation and a short pulse labeling of repair patches, intact nuclei were digested with restriction enzymes to release ϳ18% of the chromatin into soluble fragments, which are enriched (ϳ4-fold) in a constitutively transcribed gene. Upon organomercurial affinity fractionation, ϳ1.8% of the soluble chromatin remains bound in high salt (0.5 M NaCl) and is released with dithiothreitol. In normal cell chromatin, this fraction is enriched in nascent repair patches (1.5-1.8-fold) over the unbound fraction. This enrichment decreases following short chase periods with a time course similar to the loss of enhanced nuclease sensitivity of these regions (t 1 ⁄2 Ϸ 30 min). Much less enrichment of nascent repair patches is observed in the thiol-reactive fraction from XPC cells, which repair primarily the transcribed strand of active genes. These results suggest that transient nucleosome unfolding occurs during nucleotide excision repair in normal human cells, and this unfolding may require the XPC protein.

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Patterns of DNA methylation in the human and in viral genomes

Doerfler

1999

Foreign DNA in Mammalian Systems

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Chapter 12 Nucleosomes of Transcriptionally Active Chromatin: Isolation of Template-Active Nucleosomes by Affinity Chromatography

Cited by 20 publications

References 55 publications

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

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

Nucleosome Unfolding during DNA Repair in Normal and Xeroderma Pigmentosum (Group C) Human Cells

Patterns of DNA methylation in the human and in viral genomes

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