A histone octamer can step around a transcribing polymerase without leaving the template

Studitsky, Vasily M.; Clark, David; Felsenfeld, Gary

doi:10.1016/0092-8674(94)90343-3

Cited by 230 publications

(243 citation statements)

References 36 publications

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“…It has been shown that bacterial polymerases in vitro can transcribe through a nucleosome and move it backward along the DNA without complete disruption (35,36). In eukaryotic systems it has been shown that chromatin disruption complexes accompany the RNA polymerase as it moves along the DNA (37), suggesting a possible transient disruption that may not be detected in this assay.…”

Section: Discussionmentioning

confidence: 99%

Chromatin Remodeling, Measured by a Novel Real-Time Polymerase Chain Reaction Assay, Across the Proximal Promoter Region of the IL-2 Gene

Rao¹,

Procko²,

Shannon³

2001

The Journal of Immunology

183

222

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The structure of chromatin and its remodeling following activation are important aspects of the control of inducible gene transcription. The IL-2 gene is induced in a cell specific-manner in T cells following an antigenic stimulus. We show, using a novel real-time PCR assay, that significant chromatin remodeling of the IL-2 proximal promoter region occurred upon stimulation of both the murine EL-4 T cell line and primary CD4+ T cells. Chromatin remodeling appears to be limited to the first 300 bp of the proximal promoter region as measured by micrococcal nuclease and restriction enzyme accessibility. Time course studies indicated that chromatin remodeling was observed at 1.5 h postinduction and was maintained for up to 16 h. The remodeling is reversible upon removal of the stimulus. The region immediately upstream from the transcription start site, however, remains accessible for up to 16 h. Upon restimulation, remodeling occurs much more rapidly, consistent with a more rapid rise in IL-2 mRNA levels. Using a number of pharmacological inhibitors we show that remodeling is dependent on the presence of specific transcription factors, but not on the modification of histones. The development of this novel chromatin accessibility assay based on real-time PCR has allowed rapid, sensitive, and quantitative measurements on the IL-2 gene following cellular activation in both T cell lines and primary cells.

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Section: Discussionmentioning

confidence: 99%

Chromatin Remodeling, Measured by a Novel Real-Time Polymerase Chain Reaction Assay, Across the Proximal Promoter Region of the IL-2 Gene

Rao¹,

Procko²,

Shannon³

2001

The Journal of Immunology

183

222

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“…A synthetic 380-bp C-less cassette was obtained as a blunted AseI-SacI fragment from pB20A inserted at HpaI͞SacI to yield pCUP1C-less(410). pB20A was constructed by ligating an oligonucleotide containing an SP6 promoter (24) to the SmaI end of the SmaI-SacI fragment containing the C-less cassette from pCA 2 T (ref. 25; gift of M. Sawadogo, University of Texas, Houston) to yield a SacI fragment containing an SP6 promoter linked to the cassette, which was inserted into the SacI site of pB17A (with the GLN3 insert in reverse orientation relative to pB17B; ref.…”

Section: Methodsmentioning

confidence: 99%

An initiation element in the yeastCUP1promoter is recognized by RNA polymerase II in the absence of TATA box-binding protein if the DNA is negatively supercoiled

LeBlanc

Benham

Clark

2000

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

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Purified RNA polymerase II initiated transcription from the yeast CUP1 promoter fused to a C-less cassette if the DNA was negatively supercoiled. Relaxed plasmid was not transcribed. Transcription did not require addition of any other transcription factors. TATA box-binding protein (TBP) was not detectable in the polymerase preparation and the TATA box was not required. Deletion analysis of the CUP1 promoter revealed that a 25-bp element containing the initiation region was sufficient for recognition by polymerase. Two transcription start sites were mapped, one of which is identical to one of the two major start sites observed in vivo. Our observations can be accounted for by using a theoretical analysis of the probability of DNA melting within the plasmid as a function of superhelix density: the CUP1 initiation element is intrinsically unstable to superhelical stress, permitting entry of the polymerase, which then scans the DNA to locate the start site. In support of this analysis, the CUP1 promoter was sensitive to mung bean nuclease. These observations and a previous theoretical analysis of yeast genes support the idea that promoters are stress points within the DNA superhelix. The role of transcription factors might be to mark the promoter and to regulate specific melting of promoter DNA.

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“…17 The DNA was constructed with a 3′ overhang of 9 bp to facilitate ligation in future experiments and contains a site-specific cis-{Pt(NH 3 ) 2 } 2+ 1,3-d(GpTpG) cross-link. The sequence of S2-Pt is identical to that of S1-Pt, except for a shift of the platinum cross-link by 5 bp to the 5′-direction of the platinated strand.…”

Section: Design Of Dna Sequencesmentioning

confidence: 99%

Cisplatin Damage Overrides the Predefined Rotational Setting of Positioned Nucleosomes

Ober

Lippard

2007

J. Am. Chem. Soc.

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Cisplatin and carboplatin are used successfully to treat various types of cancer. The drugs target the nucleosomes of cancer cells and form intrastrand DNA cross-links that are located in the major groove. We constructed two site-specifically modified nucleosomes containing defined intrastrand cis-{Pt(NH 3 ) 2 } 2+ 1,3-d(GpTpG) cross-links. Histones from HeLa-S3 cancer cells were transferred onto synthetic DNA duplexes having nucleosome positioning sequences. The structures of these complexes were investigated by hydroxyl radical footprinting. Employing nucleosome positioning sequences allowed us to quantify the structural deviation induced by the cisplatin adduct. Our experiments demonstrate that a platinum cross-link locally overrides the rotational setting predefined in the nucleosome positioning sequence such that the lesion faces toward the histone core. Identical results were obtained for two DNA duplexes in which the sites of platination differed by approximately half a helical turn. Additionally, we determined that cisplatin unwinds nucleosomal DNA globally by approximately 24°. The intrastrand cis-{Pt(NH 3 ) 2 } 2+ 1,3-d(GpTpG) cross-links are located in an area of the nucleosome that contains locally overwound DNA in undamaged reference nucleosomes. Because most nucleosome positions in vivo are defined by the intrinsic DNA sequence, the ability of cisplatin to influence the structure of these positioned nucleosomes may be of physiological relevance.

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A histone octamer can step around a transcribing polymerase without leaving the template

Cited by 230 publications

References 36 publications

Chromatin Remodeling, Measured by a Novel Real-Time Polymerase Chain Reaction Assay, Across the Proximal Promoter Region of the IL-2 Gene

Chromatin Remodeling, Measured by a Novel Real-Time Polymerase Chain Reaction Assay, Across the Proximal Promoter Region of the IL-2 Gene

An initiation element in the yeastCUP1promoter is recognized by RNA polymerase II in the absence of TATA box-binding protein if the DNA is negatively supercoiled

Cisplatin Damage Overrides the Predefined Rotational Setting of Positioned Nucleosomes

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