Effects of Histone Tail Domains on the Rate of Transcriptional Elongation through a Nucleosome

ABSTRACT The N-terminal tail domains of the core histones play important roles in gene regulation, but the exact mechanisms through which they act are not known. Recent studies suggest that the tail domains may influence the ability of RNA polymerase to elongate through the nucleosomal DNA and, thus, that posttranslational modification of the tail domains may provide a control point for gene regulation through effects on the elongation rate. We take advantage of an experimental system that uses bacteriophage T7 RNA polymerase as a probe for aspects of nucleosome transcription that are dominated by the properties of nucleosomes themselves. With this system, experiments can analyze the synchronous, real-time, single-passage transcription on the nucleosomal template. Here, we use this system to directly test the hypothesis that the tail domains may influence the “elongatability” of nucleosomal DNA and to identify which of the tail domains may contribute to this. The results show that the tail domains strongly influence the rate of elongation and suggest that the effect is dominated by the N-terminal domains of the (H3-H4)2 tetramer. They further imply that tail-mediated octamer transfer is not essential for elongation through the nucleosome. Acetylation of the tail domains leads to effects on elongation that are similar to those arising from complete removal of the tail domains.

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Nucleosomal Fluctuations Govern the Transcription Dynamics of RNA Polymerase II

Science ◽

10.1126/science.1172926 ◽

2009 ◽

Vol 325 (5940) ◽

pp. 626-628 ◽

Cited By ~ 243

Author(s):

Courtney Hodges ◽

Lacramioara Bintu ◽

Lucyna Lubkowska ◽

Mikhail Kashlev ◽

Carlos Bustamante

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Optical Tweezers ◽

Direct Evidence ◽

Eukaryotic Transcription ◽

Pol Ii ◽

The Core ◽

Nucleosomal Dna ◽

Dna Loop ◽

Core Histones

RNA polymerase II (Pol II) must overcome the barriers imposed by nucleosomes during transcription elongation. We have developed an optical tweezers assay to follow individual Pol II complexes as they transcribe nucleosomal DNA. Our results indicate that the nucleosome behaves as a fluctuating barrier that locally increases pause density, slows pause recovery, and reduces the apparent pause-free velocity of Pol II. The polymerase, rather than actively separating DNA from histones, functions instead as a ratchet that rectifies nucleosomal fluctuations. We also obtained direct evidence that transcription through a nucleosome involves transfer of the core histones behind the transcribing polymerase via a transient DNA loop. The interplay between polymerase dynamics and nucleosome fluctuations provides a physical basis for the regulation of eukaryotic transcription.

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Conformational changes induced by salt in complexes of histones and superhelical nuclear DNA

Journal of Cell Science ◽

10.1242/jcs.50.1.199 ◽

1981 ◽

Vol 50 (1) ◽

pp. 199-208

Author(s):

J.M. Levin ◽

P.R. Cook

Keyword(s):

Free Energy ◽

Rna Polymerase ◽

Conformational Changes ◽

Salt Concentration ◽

Nuclear Dna ◽

The Core ◽

Ionic Detergent ◽

Core Histones ◽

Intercalating Dye ◽

Negative Supercoiling

When HeLa cells are lysed in solutions containing a non-ionic detergent and 0.75 M-NaCl, structures are released that retain many of the morphological features of nuclei. These nucleoids contain all the nuclear DNA, RNA and the ‘core’ histones, but few other proteins characteristic of chromatin. Their DNA is intact. The core histones dissociate on raising the salt concentration. We have probed the structure of nucleoid-histone complexes using the intercalating dye, ethidium, or the RNA polymerase of Escherichia coli. Both have a higher affinity for superhelical DNA than they do for relaxed DNA. The binding of ethidium is measured fluorometrically, and using this probe we find that the DNA of nucleoids containing all the core histones behaves as if it were supercoiled slightly positively. As the salt concentration is increased, free energy characteristic of negative supercoiling appears between 0.92 M and 0.95 M-NaCl. This transition, which is reversible in the presence of the arginine-rich histones, occurs without dissociation of these histones from the DNA and so must reflect a conformational change in the complex. In contrast to the results with ethidium, we find that RNA polymerase can detect the presence of some negative free energy of supercoiling in nucleoids containing the core histones. The transformations of the free energy that can assist the binding of ethidium and RNA polymerase are discussed.

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Evidence for Eviction and Rapid Deposition of Histones upon Transcriptional Elongation by RNA Polymerase II

Molecular and Cellular Biology ◽

10.1128/mcb.24.23.10111-10117.2004 ◽

2004 ◽

Vol 24 (23) ◽

pp. 10111-10117 ◽

Cited By ~ 250

Author(s):

Marc A. Schwabish ◽

Kevin Struhl

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Dynamic Equilibrium ◽

Yeast Genome ◽

Transcriptional Elongation ◽

Coding Region ◽

Pol Ii ◽

Core Histones ◽

Histone Exchange

ABSTRACT Biochemical experiments indicate that transcriptional elongation by RNA polymerase II (Pol II) is inhibited by nucleosomes and hence requires chromatin-modifying activities. Here, we examine the fate of histones upon passage of elongating Pol II in vivo. Histone density throughout the entire Saccharomyces cerevisiae GAL10 coding region is inversely correlated with Pol II association and transcriptional activity, suggesting that the elongating Pol II machinery efficiently evicts core histones from the DNA. Furthermore, new histones appear to be deposited onto DNA less than 1 min after passage of Pol II. Transcription-dependent deposition of histones requires the FACT complex that travels with elongating Pol II. Our results suggest that Pol II transcription generates a highly dynamic equilibrium of histone eviction and histone deposition and that there is significant histone exchange throughout most of the yeast genome within a single cell cycle.

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The amino-terminal tails of the core histones and the translational position of the TATA box determine TBP/TFIIA association with nucleosomal DNA

Nucleic Acids Research ◽

10.1093/nar/23.22.4557 ◽

1995 ◽

Vol 23 (22) ◽

pp. 4557-4564 ◽

Cited By ~ 61

Author(s):

James S. Godde ◽

Yoshibiro Nakatani ◽

Alan P. Wolffe

Keyword(s):

Tata Box ◽

Amino Terminal ◽

The Core ◽

Nucleosomal Dna ◽

Core Histones

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Identification and Characterization of the Genes Encoding the Core Histones and Histone Variants of Neurospora crassa

Genetics ◽

10.1093/genetics/160.3.961 ◽

2002 ◽

Vol 160 (3) ◽

pp. 961-973 ◽

Cited By ~ 1

Author(s):

Shan M Hays ◽

Johanna Swanson ◽

Eric U Selker

Keyword(s):

Neurospora Crassa ◽

Histone Variants ◽

Null Alleles ◽

Histone Genes ◽

Histone H4 ◽

Coding Regions ◽

The Core ◽

Genomic Arrangement ◽

Genes Encoding ◽

Core Histones

Abstract We have identified and characterized the complete complement of genes encoding the core histones of Neurospora crassa. In addition to the previously identified pair of genes that encode histones H3 and H4 (hH3 and hH4-1), we identified a second histone H4 gene (hH4-2), a divergently transcribed pair of genes that encode H2A and H2B (hH2A and hH2B), a homolog of the F/Z family of H2A variants (hH2Az), a homolog of the H3 variant CSE4 from Saccharomyces cerevisiae (hH3v), and a highly diverged H4 variant (hH4v) not described in other species. The hH4-1 and hH4-2 genes, which are 96% identical in their coding regions and encode identical proteins, were inactivated independently. Strains with inactivating mutations in either gene were phenotypically wild type, in terms of growth rates and fertility, but the double mutants were inviable. As expected, we were unable to isolate null alleles of hH2A, hH2B, or hH3. The genomic arrangement of the histone and histone variant genes was determined. hH2Az and the hH3-hH4-1 gene pair are on LG IIR, with hH2Az centromere-proximal to hH3-hH4-1 and hH3 centromere-proximal to hH4-1. hH3v and hH4-2 are on LG IIIR with hH3v centromere-proximal to hH4-2. hH4v is on LG IVR and the hH2A-hH2B pair is located immediately right of the LG VII centromere, with hH2A centromere-proximal to hH2B. Except for the centromere-distal gene in the pairs, all of the histone genes are transcribed toward the centromere. Phylogenetic analysis of the N. crassa histone genes places them in the Euascomycota lineage. In contrast to the general case in eukaryotes, histone genes in euascomycetes are few in number and contain introns. This may be a reflection of the evolution of the RIP (repeat-induced point mutation) and MIP (methylation induced premeiotically) processes that detect sizable duplications and silence associated genes.

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Persistent Interactions of Core Histone Tails with Nucleosomal DNA following Acetylation and Transcription Factor Binding

Molecular and Cellular Biology ◽

10.1128/mcb.18.11.6293 ◽

1998 ◽

Vol 18 (11) ◽

pp. 6293-6304 ◽

Cited By ~ 96

Author(s):

Vesco Mutskov ◽

Delphine Gerber ◽

Dimitri Angelov ◽

Juan Ausio ◽

Jerry Workman ◽

...

Keyword(s):

Transcription Factor ◽

Transcription Factors ◽

Molecular Biology ◽

Binding Sites ◽

Cross Linking ◽

Uv Laser ◽

Histone Tail ◽

Histone Tails ◽

Nucleosomal Dna ◽

Core Histones

ABSTRACT In this study, we examined the effect of acetylation of the NH2 tails of core histones on their binding to nucleosomal DNA in the absence or presence of bound transcription factors. To do this, we used a novel UV laser-induced protein-DNA cross-linking technique, combined with immunochemical and molecular biology approaches. Nucleosomes containing one or five GAL4 binding sites were reconstituted with hypoacetylated or hyperacetylated core histones. Within these reconstituted particles, UV laser-induced histone-DNA cross-linking was found to occur only via the nonstructured histone tails and thus presented a unique tool for studying histone tail interactions with nucleosomal DNA. Importantly, these studies demonstrated that the NH2 tails were not released from nucleosomal DNA upon histone acetylation, although some weakening of their interactions was observed at elevated ionic strengths. Moreover, the binding of up to five GAL4-AH dimers to nucleosomes occupying the central 90 bp occurred without displacement of the histone NH2 tails from DNA. GAL4-AH binding perturbed the interaction of each histone tail with nucleosomal DNA to different degrees. However, in all cases, greater than 50% of the interactions between the histone tails and DNA was retained upon GAL4-AH binding, even if the tails were highly acetylated. These data illustrate an interaction of acetylated or nonacetylated histone tails with DNA that persists in the presence of simultaneously bound transcription factors.

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