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The yeast histone chaperone hif1p functions with RNA in nucleosome assembly.

Knapp AR, Wang H, Parthun MR - PLoS ONE (2014)

Bottom Line: Analysis of partially purified fractions indicated that Hif1p-dependent chromatin assembly activity resided in RNA rather than protein.Fractionation of isolated RNA indicated that the chromatin assembly activity did not simply purify with bulk RNA.These results suggest that specific RNA species may function in concert with histone chaperones to assemble chromatin.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biochemistry, The Ohio State University, Columbus, Ohio, United States of America.

ABSTRACT

Background: Hif1p is an H3/H4-specific histone chaperone that associates with the nuclear form of the Hat1p/Hat2p complex (NuB4 complex) in the yeast Saccharomyces cerevisiae. While not capable of depositing histones onto DNA on its own, Hif1p can act in conjunction with a yeast cytosolic extract to assemble nucleosomes onto a relaxed circular plasmid.

Results: To identify the factor(s) that function with Hif1p to carry out chromatin assembly, multiple steps of column chromatography were carried out to fractionate the yeast cytosolic extract. Analysis of partially purified fractions indicated that Hif1p-dependent chromatin assembly activity resided in RNA rather than protein. Fractionation of isolated RNA indicated that the chromatin assembly activity did not simply purify with bulk RNA. In addition, the RNA-mediated chromatin assembly activity was blocked by mutations in the human homolog of Hif1p, sNASP, that prevent the association of this histone chaperone with histone H3 and H4 without altering its electrostatic properties.

Conclusions: These results suggest that specific RNA species may function in concert with histone chaperones to assemble chromatin.

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Related in: MedlinePlus

RNA-based chromatin assembly factor localizes to a discreet size range.The material that flowed through the DEAE, carboxymethyl and heparin columns was deproteinized and resolved on a polyacrylamide/urea gel. Segments were cut out of the gel and RNA was eluted (numbered lanes). Recovered RNA was either resolved by polyacrylamide:urea electrophoresis and visualized with Sybr Gold (top) or assayed for chromatin assembly activity with a relaxed circular plasmid, core histones and in the absence or presence of rHif1p, as indicated (bottom). Supercoiled and relaxed plasmid is indicated (S and R, respectively).
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pone-0100299-g006: RNA-based chromatin assembly factor localizes to a discreet size range.The material that flowed through the DEAE, carboxymethyl and heparin columns was deproteinized and resolved on a polyacrylamide/urea gel. Segments were cut out of the gel and RNA was eluted (numbered lanes). Recovered RNA was either resolved by polyacrylamide:urea electrophoresis and visualized with Sybr Gold (top) or assayed for chromatin assembly activity with a relaxed circular plasmid, core histones and in the absence or presence of rHif1p, as indicated (bottom). Supercoiled and relaxed plasmid is indicated (S and R, respectively).

Mentions: To further test whether the chromatin assembly activity was due to the presence of specific RNA species or whether it was attributable to a bulk RNA effect, the isolated RNA that flowed through the DEAE, CM and heparin columns was resolved by polyacrylamide:urea electrophoresis. The gel lane was then cut into a series of 1.0 cm sections and the RNA was eluted from each. Aliquots from each pool of RNA were then either re-run on a gel or assayed for chromatin assembly activity. As seen in Figure 6, the chromatin assembly activity was found in the first three segment containing RNA molecules longer than ∼700 bases. Importantly, the level of chromatin assembly activity did not correlate with bulk RNA concentration as would be predicted if the RNA was functioning as a non-specific polyanion.


The yeast histone chaperone hif1p functions with RNA in nucleosome assembly.

Knapp AR, Wang H, Parthun MR - PLoS ONE (2014)

RNA-based chromatin assembly factor localizes to a discreet size range.The material that flowed through the DEAE, carboxymethyl and heparin columns was deproteinized and resolved on a polyacrylamide/urea gel. Segments were cut out of the gel and RNA was eluted (numbered lanes). Recovered RNA was either resolved by polyacrylamide:urea electrophoresis and visualized with Sybr Gold (top) or assayed for chromatin assembly activity with a relaxed circular plasmid, core histones and in the absence or presence of rHif1p, as indicated (bottom). Supercoiled and relaxed plasmid is indicated (S and R, respectively).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4114455&req=5

pone-0100299-g006: RNA-based chromatin assembly factor localizes to a discreet size range.The material that flowed through the DEAE, carboxymethyl and heparin columns was deproteinized and resolved on a polyacrylamide/urea gel. Segments were cut out of the gel and RNA was eluted (numbered lanes). Recovered RNA was either resolved by polyacrylamide:urea electrophoresis and visualized with Sybr Gold (top) or assayed for chromatin assembly activity with a relaxed circular plasmid, core histones and in the absence or presence of rHif1p, as indicated (bottom). Supercoiled and relaxed plasmid is indicated (S and R, respectively).
Mentions: To further test whether the chromatin assembly activity was due to the presence of specific RNA species or whether it was attributable to a bulk RNA effect, the isolated RNA that flowed through the DEAE, CM and heparin columns was resolved by polyacrylamide:urea electrophoresis. The gel lane was then cut into a series of 1.0 cm sections and the RNA was eluted from each. Aliquots from each pool of RNA were then either re-run on a gel or assayed for chromatin assembly activity. As seen in Figure 6, the chromatin assembly activity was found in the first three segment containing RNA molecules longer than ∼700 bases. Importantly, the level of chromatin assembly activity did not correlate with bulk RNA concentration as would be predicted if the RNA was functioning as a non-specific polyanion.

Bottom Line: Analysis of partially purified fractions indicated that Hif1p-dependent chromatin assembly activity resided in RNA rather than protein.Fractionation of isolated RNA indicated that the chromatin assembly activity did not simply purify with bulk RNA.These results suggest that specific RNA species may function in concert with histone chaperones to assemble chromatin.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biochemistry, The Ohio State University, Columbus, Ohio, United States of America.

ABSTRACT

Background: Hif1p is an H3/H4-specific histone chaperone that associates with the nuclear form of the Hat1p/Hat2p complex (NuB4 complex) in the yeast Saccharomyces cerevisiae. While not capable of depositing histones onto DNA on its own, Hif1p can act in conjunction with a yeast cytosolic extract to assemble nucleosomes onto a relaxed circular plasmid.

Results: To identify the factor(s) that function with Hif1p to carry out chromatin assembly, multiple steps of column chromatography were carried out to fractionate the yeast cytosolic extract. Analysis of partially purified fractions indicated that Hif1p-dependent chromatin assembly activity resided in RNA rather than protein. Fractionation of isolated RNA indicated that the chromatin assembly activity did not simply purify with bulk RNA. In addition, the RNA-mediated chromatin assembly activity was blocked by mutations in the human homolog of Hif1p, sNASP, that prevent the association of this histone chaperone with histone H3 and H4 without altering its electrostatic properties.

Conclusions: These results suggest that specific RNA species may function in concert with histone chaperones to assemble chromatin.

Show MeSH
Related in: MedlinePlus