Nucleosome Positioning on Chicken and Human Globin Gene Promoters in Vitro

Yenidünya, Ali Fazıl; Davey, Colin S.; Clark, David; Felsenfeld, Gary; Allan, James

doi:10.1006/jmbi.1994.1243

Cited by 32 publications

(42 citation statements)

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“…The unique XbaI site used in the mapping experiments shown here is located in the TRP1 ORF. (B) Schematic diagram of the monomer extension method for mapping the positions of nucleosomes (41). The approach is to obtain DNA from nucleosome core particles (mononucleosomes or monomers) and use this as primer in a primer extension reaction; since both strands of nucleosomal DNA can act as primers, a single-stranded template must be used (otherwise two sets of extension products will be obtained).…”

Section: Resultsmentioning

confidence: 99%

“…Markers (lanes 1, 2, and 3) were as described in the legend for undergoes structural transitions similar to those of HIS3 plasmid chromatin. However, the monomer extension method cannot be applied to single-copy genes in the context of the entire genome because the huge excess of nucleosomes derived from the rest of the genome results in nonspecific annealing to template DNA, contributing too much background (41). Plasmid-borne HIS3 can be purified away from the rest of the genome, and the signal is improved by the high copy number of TA-HIS3.…”

Section: Resultsmentioning

confidence: 99%

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Activation of Saccharomyces cerevisiae HIS3 Results in Gcn4p-Dependent, SWI/SNF-Dependent Mobilization of Nucleosomes over the Entire Gene

Kim

McLaughlin

Lindstrom

et al. 2006

Molecular and Cellular Biology

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The effects of transcriptional activation on the chromatin structure of the Saccharomyces cerevisiae HIS3 gene were addressed by mapping the precise positions of nucleosomes in uninduced and induced chromatin. In the absence of the Gcn4p activator, the HIS3 gene is organized into a predominant nucleosomal array. In wild-type chromatin, this array is disrupted, and several alternative overlapping nucleosomal arrays are formed. The disruption of the predominant array also requires the SWI/SNF remodeling machine, indicating that the SWI/SNF complex plays an important role in nucleosome mobilization over the entire HIS3 gene. The Isw1 remodeling complex plays a more subtle role in determining nucleosome positions on HIS3, favoring positions different from those preferred by the SWI/SNF complex. Both the SWI/SNF and Isw1 complexes are constitutively present in HIS3 chromatin, although Isw1 tends to be excluded from the HIS3 promoter. Despite the apparent disorder of HIS3 chromatin generated by the formation of multiple nucleosomal arrays, nucleosome density profiles indicate that some long-range order is always present. We propose that Gcn4p stimulates nucleosome mobilization over the entire HIS3 gene by the SWI/SNF complex. We suggest that the net effect of interplay among remodeling machines at HIS3 is to create a highly dynamic chromatin structure.The central role of chromatin structure in gene regulation is currently the subject of intense interest. Much has been learned about the ATP-dependent chromatin remodeling machines, which use the free energy of ATP hydrolysis to move nucleosomes along DNA and alter nucleosome conformation (31), and the histone-modifying enzymes and their complexes. There is also a great deal of information available concerning changes in chromatin structure occurring at various gene promoters (3,20). Because most of the obvious changes in chromatin structure occur at gene promoters, emphasis has been placed on these events, which are clearly of major importance. However, in our high-resolution studies of the chromatin structure of the CUP1 and HIS3 genes of Saccharomyces cerevisiae, we have obtained evidence pointing to more widespread changes in chromatin structure resulting from induction; these changes involve the entire gene and flanking sequences (9, 29). Our observations indicate that, at least for these genes, chromatin remodeling is not confined to the promoter but occurs on the scale of a chromatin domain.It is now apparent that ATP-dependent chromatin remodeling machines can be grouped into several functional classes (2). One class includes the SWI/SNF and RSC complexes, which are capable of mobilizing nucleosomes and driving a conformational change in the nucleosome to create the remodeled state (24,31). A second class is defined by the ISWI group of complexes, exemplified in budding yeast by the Isw1 and Isw2 complexes, which are capable of mobilizing nucleosomes to create arrays with characteristic spacing but cannot remodel nucleosome structure (19,39). A third class of com...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Activation of Saccharomyces cerevisiae HIS3 Results in Gcn4p-Dependent, SWI/SNF-Dependent Mobilization of Nucleosomes over the Entire Gene

Kim

McLaughlin

Lindstrom

et al. 2006

Molecular and Cellular Biology

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show abstract

“…Core particle DNA was extracted, purified from 3% agarose gels, and end labeled with T4 kinase. Core DNA was denatured with alkali, annealed with excess template, and extended with Klenow enzyme, in the presence or absence of a restriction enzyme (61). With pGEM-TAC(ϩ) as template, BamHI, SapI, XcmI, Bsu36I, and DraIII were used.…”

Section: Purification Of Minichromosomesmentioning

confidence: 99%

Remodeling of YeastCUP1Chromatin Involves Activator-Dependent Repositioning of Nucleosomes over the Entire Gene and Flanking Sequences

Shen

LeBlanc

Alfieri

et al. 2001

Molecular and Cellular Biology

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The yeast CUP1 gene is activated by the copper-dependent binding of the transcriptional activator, Ace1p. An episome containing transcriptionally active or inactive CUP1 was purified in its native chromatin structure from yeast cells. The amount of RNA polymerase II on CUP1 in the purified episomes correlated with its transcriptional activity in vivo. Chromatin structures were examined by using the monomer extension technique to map translational positions of nucleosomes. The chromatin structure of an episome containing inactive CUP1 isolated from ace1⌬ cells is organized into clusters of overlapping nucleosome positions separated by linkers. Novel nucleosome positions that include the linkers are occupied in the presence of Ace1p. Repositioning was observed over the entire CUP1 gene and its flanking regions, possibly over the entire episome. Mutation of the TATA boxes to prevent transcription did not prevent repositioning, implicating a chromatin remodeling activity recruited by Ace1p. These observations provide direct evidence in vivo for the nucleosome sliding mechanism proposed for remodeling complexes in vitro and indicate that remodeling is not restricted to the promoter but occurs over a chromatin domain including CUP1 and its flanking sequences.

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“…Sequences that position nucleosomes within ␤-globin loci have been reported. 51 The Oct-1 site may be more important than the Gata-1 site for elevating expression levels directed by the intron-2 enhancer in this scenario, but the AT sequences alone could be sufficient to establish weaker unanchored nucleosome positioning and permit detectable expression at all transgene integration sites. …”

Section: Mutant Atr Constructs Express Detectable Levels At Ectopic Smentioning

confidence: 99%

LCR-regulated transgene expression levels depend on the Oct-1 site in the AT-rich region of β-globin intron-2

Bharadwaj

Trainor

Pasceri

et al. 2003

Blood

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Human ␤-globin transgenes regulated by the locus control region (LCR) express at all integration sites in transgenic mice. For such LCR activity at ectopic sites, the 5HS3 element requires the presence of the AT-rich region (ATR) in ␤-globin intron-2. Here, we examine the dependence of 5HS3 LCR activity on transcription factor binding sites in the ATR. In vitro DNaseI footprint analysis and electrophoretic mobility shift assays of the ATR identified an inverted double Gata-1 site composed of 2 noncanonical sequences (GATT and GATG) and an Oct-1 consensus site. Mutant Oct-1, Gata-1, or double mutant sites were created in the ATR of the BGT50 construct composed of a 5HS3 ␤/␥-globin hybrid transgene. Transgenes with double mutant sites expressed at all sites of integration, but mean expression levels in transgenic mice were reduced from 64% per copy (BGT50) to 37% (P < .05). Mutation of the inverted double Gata-1 site had no effect at 61% per copy expression levels. In contrast, mutation of the Oct-1 site alone reduced per-copy expression levels to 31% (P < .05). We conclude that the ability of 5HS3 to activate expression from all transgene integration sites is dependent on sequences in the ATR that are not bound at high affinity by transcription factors. In addition, the Oct-1 site in the ATR is required for high-level 5HS3 ␤/␥-globin transgene expression and should be retained in LCR␤-globin expression cassettes designed for gene therapy. (Blood. 2003;

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Nucleosome Positioning on Chicken and Human Globin Gene Promoters in Vitro

Cited by 32 publications

References 0 publications

Activation of Saccharomyces cerevisiae HIS3 Results in Gcn4p-Dependent, SWI/SNF-Dependent Mobilization of Nucleosomes over the Entire Gene

Activation of Saccharomyces cerevisiae HIS3 Results in Gcn4p-Dependent, SWI/SNF-Dependent Mobilization of Nucleosomes over the Entire Gene

Remodeling of YeastCUP1Chromatin Involves Activator-Dependent Repositioning of Nucleosomes over the Entire Gene and Flanking Sequences

LCR-regulated transgene expression levels depend on the Oct-1 site in the AT-rich region of β-globin intron-2

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