Disruption of Reconstituted Nucleosomes

Godde, James S.; Wolffe, Alan P.

doi:10.1074/jbc.270.46.27399

Cited by 41 publications

(17 citation statements)

References 41 publications

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“…Differences between the experiments of Lee et al (22) and our work include the use of high concentrations of MgCl 2 and more dilute nucleosome concentrations by Lee et al (22) (in our study an approximately 100-fold excess of nucleosome cores to labeled DNA was used for exchange reconstitutions, whereas Lee et al [22] used only a 5-fold excess). Both of these factors have been shown to lead to disruption of histone-DNA contacts (3,11,15). An additional factor which could explain the differing results is the use of a slightly higher TFIIIA concentration in our study than in that of Lee et al (22).…”

Section: Discussioncontrasting

confidence: 55%

Nucleosome Translational Position, Not Histone Acetylation, Determines TFIIIA Binding to Nucleosomal Xenopus laevis 5S rRNA Genes

Howe

Ausió

1998

Molecular and Cellular Biology

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We sought to study the binding constraints placed on the nine-zinc-finger protein transcription factor IIIA (TFIIIA) by a histone octamer. To this end, five overlapping fragments of the Xenopus laevis oocyte and somatic 5S rRNA genes were reconstituted into nucleosomes, and it was subsequently shown that nucleosome translational positioning is a major determinant of the binding of TFIIIA to the 5S rRNA genes. Furthermore, it was found that histone acetylation cannot override the TFIIIA binding constraints imposed by unfavorable translational positions.Xenopus laevis produces two major types of 5S rRNA: the somatic type is synthesized in most cell types, whereas the oocyte type is synthesized during early oogenesis, during embryogenesis, and in certain tissue culture cell lines (14,47). Each 5S rRNA type is transcribed from a distinct multigene family, and considerable research has focused on understanding the differential expression of these genes. One hypothesis suggests that the transcription complexes which form on the oocyte genes are relatively unstable compared to those of the somatic counterparts. As a result of this, the oocyte genes are transcribed only when transcription factor IIIA (TFIIIA) levels are relatively high, as is the case during oogenesis. In somatic cells, in which TFIIIA levels are much lower, the transcription complexes dissociate from the oocyte gene, allowing the subsequent assembly of repressive nucleosome structures which preclude further factor binding (47). Conflicting with this hypothesis are results which show that, at least in vitro, the RNA polymerase III transcription complexes, once formed, have similar stabilities on both the oocyte and somatic genes (35). Thus, at this time, the reasons for the differential transcription of these two gene families in X. laevis are not fully understood.The results of 5S rRNA gene transcription studies, performed with both native and reconstituted chromatin as templates, have been conflicting. In one such study it was demonstrated that chromatin isolated from a Xenopus kidney cell line can serve as a template for transcription of the oocyte gene after the removal of histone H1 (34). In addition, it has been shown that incorporation of a somatic histone H1 variant into chromatin during embryogenesis results in specific repression of TFIIIA-activated oocyte 5S rRNA transcription (8). Furthermore, histone H1 has been shown to specifically repress transcription of oocyte genes in reconstituted chromatin while leaving the corresponding somatic genes unaffected (40). These results suggest that it is the presence of histone H1 which is responsible for the repression of oocyte transcription. This is not surprising considering that histone H1 is thought of as a repressor of transcription, although the exact mechanism of this repression is not known. In contrast to the abovementioned work, studies with chromatin reconstituted in the absence of linker histones have shown that the presence of nucleosome core particles alone is sufficient to repress oocyt...

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Section: Discussioncontrasting

confidence: 55%

Nucleosome Translational Position, Not Histone Acetylation, Determines TFIIIA Binding to Nucleosomal Xenopus laevis 5S rRNA Genes

Howe

Ausió

1998

Molecular and Cellular Biology

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“…The slightly higher affinity of HNF3 for the eG site on free DNA was observed in previous footprint titration studies, with the template concentration below the dissociation constant, as was DNase I hypersensitivity within the HNF3 footprints (Zaret and Stevens 1995). The nucleosome concentrations used in all experiments herein, 2 ng/µl, is within that required for nucleosome stability in vitro (Godde and Wolffe 1995). With a 4 nM concentration of dinucleosomes, 30 nM of HNF3 resulted in simultaneous occupancy of both the eG and eH sites and DNase hypersensitivity, but at both sites the footprints were smaller than that observed on free DNA ( Fig.…”

Section: Hnf3␣ Binds Its Target Sites On Dinucleosome Templatesmentioning

confidence: 71%

Nucleosome positioning by the winged helix transcription factor HNF3

Shim¹,

Woodcock²,

Zaret³

1998

Genes Dev.

120

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Nucleosome positioning at genetic regulatory sequences is not well understood. The transcriptional enhancer of the mouse serum albumin gene is active in liver, where regulatory factors occupy their target sites on three nucleosome-like particles designated N1, N2, and N3. The winged helix transcription factor HNF3 binds to two sites near the center of the N1 particle. We created dinucleosome templates using the albumin enhancer sequence and found that site-specific binding of HNF3 protein resulted in nucleosome positioning in vitro similar to that seen in liver nuclei. Thus, binding of a transcription factor can position an underlying nucleosome core.

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“…We note that in our nucleosome preparations a small fraction of the substrate exhibited much more rapid cleavage kinetics than the bulk. Given the well known propensity of nucleosomes to undergo disproportionation or dissociation, especially at low concentrations (55,56), we believe that the rapidly digesting component in our experiments represents a non-nucleosomal contaminant that can dominate the kinetic profile of a more slowly digesting component at low enzyme concentrations. This is a critical issue as standard enzyme kinetic assays typically examine the first few percent of substrate processed, something clearly impossible for nucleosome samples.…”

Section: Discussionmentioning

confidence: 98%

Uracil DNA Glycosylase Activity on Nucleosomal DNA Depends on Rotational Orientation of Targets

Cole¹,

Tabor-Godwin²,

Hayes

2010

Journal of Biological Chemistry

101

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The activity of uracil DNA glycosylases (UDGs), which recognize and excise uracil bases from DNA, has been well characterized on naked DNA substrates but less is known about activity in chromatin. We therefore prepared a set of model nucleosome substrates in which single thymidine residues were replaced with uracil at specific locations and a second set of nucleosomes in which uracils were randomly substituted for all thymidines. We found that UDG efficiently removes uracil from internal locations in the nucleosome where the DNA backbone is oriented away from the surface of the histone octamer, without significant disruption of histone-DNA interactions. However, uracils at sites oriented toward the histone octamer surface were excised at much slower rates, consistent with a mechanism requiring spontaneous DNA unwrapping from the nucleosome. In contrast to the nucleosome core, UDG activity on DNA outside the core DNA region was similar to that of naked DNA. Association of linker histone reduced activity of UDG at selected sites near where the globular domain of H1 is proposed to bind to the nucleosome as well as within the extra-core DNA. Our results indicate that some sites within the nucleosome core and the extra-core (linker) DNA regions represent hot spots for repair that could influence critical biological processes.

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Disruption of Reconstituted Nucleosomes

Cited by 41 publications

References 41 publications

Nucleosome Translational Position, Not Histone Acetylation, Determines TFIIIA Binding to Nucleosomal Xenopus laevis 5S rRNA Genes

Nucleosome Translational Position, Not Histone Acetylation, Determines TFIIIA Binding to Nucleosomal Xenopus laevis 5S rRNA Genes

Nucleosome positioning by the winged helix transcription factor HNF3

Uracil DNA Glycosylase Activity on Nucleosomal DNA Depends on Rotational Orientation of Targets

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