The Nonessential H2A N-Terminal Tail Can Function as an Essential Charge Patch on the H2A.Z Variant N-Terminal Tail

Ren, Qinghu; Gorovsky, Martin A.

doi:10.1128/mcb.23.8.2778-2789.2003

Cited by 33 publications

(44 citation statements)

References 92 publications

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“…Strains CU428, CU427, and B2086 were provided by P. J. Bruns (Cornell University). Major histone H2A (H2A.X and H2A.1) germ line double knockout heterokaryon strains G4A1F14A and G4B1G6A and all mutant strains were generated as previously described (62). For studies of vegetative growth, Tetrahymena cells were grown in super proteose peptone (SPP) medium (31) containing 1% proteose peptone (1ϫ SPP).…”

Section: Methodsmentioning

confidence: 99%

“…For conjugation, two strains of different mating types were washed, starved (15 to 24 h, without shaking at 30°C), and mated in 10 mM Tris-HCl (pH 7.5) as previously described (3). Major H2A genes (HTAX and HTA1) germ line double knockout heterokaryons and sitedirected mutagenesis were generated and performed as described (62).…”

Section: Methodsmentioning

confidence: 99%

“…Acid-urea polyacrylamide gel electrophoresis (AU-PAGE) was performed as described previously (5,62).…”

Section: Methodsmentioning

confidence: 99%

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Phosphorylation of the SQ H2A.X Motif Is Required for Proper Meiosis and Mitosis in Tetrahymena thermophila

Song

Gjoneska

Ren

et al. 2007

Molecular and Cellular Biology

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Phosphorylation of the C terminus SQ motif that defines H2A.X variants is required for efficient DNA double-strand break (DSB) repair in diverse organisms but has not been studied in ciliated protozoa. Tetrahymena H2A.X is one of two similarly expressed major H2As, thereby differing both from mammals, where H2A.X is a quantitatively minor component, and from Saccharomyces cerevisiae where it is the only type of major H2A. Tetrahymena H2A.X is phosphorylated in the SQ motif in both the mitotic micronucleus and the amitotic macronucleus in response to DSBs induced by chemical agents and in the micronucleus during prophase of meiosis, which occurs in the absence of a synaptonemal complex. H2A.X is phosphorylated when programmed DNA rearrangements occur in developing macronuclei, as for immunoglobulin gene rearrangements in mammals, but not during the DNA fragmentation that accompanies breakdown of the parental macronucleus during conjugation, correcting the previous interpretation that this process is apoptosis-like. Using strains containing a mutated (S134A) SQ motif, we demonstrate that phosphorylation of this motif is important for Tetrahymena cells to recover from exogenous DNA damage and is required for normal micronuclear meiosis and mitosis and, to a lesser extent, for normal amitotic macronuclear division; its absence, while not lethal, leads to the accumulation of DSBs in both micro-and macronuclei. These results demonstrate multiple roles of H2A.X phosphorylation in maintaining genomic integrity in different phases of the Tetrahymena life cycle.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Phosphorylation of the SQ H2A.X Motif Is Required for Proper Meiosis and Mitosis in Tetrahymena thermophila

Song

Gjoneska

Ren

et al. 2007

Molecular and Cellular Biology

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“…The X-ray crystal structure of nucleosomes containing H2A.Z has been determined and, together with the results of biochemical experiments, argues that H2A.Z alters the physical properties of the chromatin template (7). Histone H2A.Z is itself subject to posttranslational modifications, including acetylation, ubiquitylation, and sumoylation, and these may alter its chromatin association, exchange dynamics, structure, and function (8,32,39,41,43,66,81,84,95). H2A.Z is broadly but nonuniformly distributed throughout the chromosomes and is deposited by specific remodeling complexes, such as the yeast SWR1 complex (SWR1) (3,45,51,60,67).…”

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confidence: 99%

Histone Variant H2A.Z and RNA Polymerase II Transcription Elongation

Santisteban

Hang

Smith

2011

Molecular and Cellular Biology

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Nucleosomes containing histone variant H2A.Z (Htz1) serve to poise quiescent genes for activation and transcriptional initiation. However, little is known about their role in transcription elongation. Here we show that dominant mutations in the elongation genes SPT5 and SPT16 suppress the hypersensitivity of htz1⌬ strains to drugs that inhibit elongation, indicating that Htz1 functions at the level of transcription elongation. Direct kinetic measurements of RNA polymerase II (Pol II) movement across the 9.5-kb GAL10p-VPS13 gene revealed that the elongation rate of polymerase is 24% slower in the absence of Htz1. We provide evidence for two nonexclusive mechanisms. First, we observed that both the phospho-Ser2 levels in the elongating isoform of Pol II and the loading of Spt5 and Elongator over the GAL1 open reading frame (ORF) depend on Htz1. Second, in the absence of Htz1, the density of nucleosome occupancy is increased over the GAL10p-VPS13 ORF and the chromatin is refractory to remodeling during active transcription. These results establish a mechanistic role for Htz1 in transcription elongation and suggest that Htz1-containing nucleosomes facilitate Pol II passage by affecting the correct assembly and modification status of Pol II elongation complexes and by favoring efficient nucleosome remodeling over the gene.Transcription by RNA polymerase II (Pol II) takes place on a compositionally and topologically complex chromatin template at multiple steps, including promoter activation, transcription initiation, elongation, and termination. A number of mechanisms facilitate the initiation of gene transcription on chromatin templates. Two of the most prominent of these are the remodeling of nucleosomes by DNA-dependent ATPase complexes and the covalent posttranslational modification of histone proteins (20,90). At a minimum, these pathways are thought to act by establishing a permissive chromatin structure at gene promoters, allowing access of site-specific transcription factors and the subsequent recruitment of polymerase and the general factors (25,69).

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“…The N-terminal region 39 amino acids are important for DNA-histone and histone-histone interactions within and/or between nucleosomes and for interaction with nonhistone proteins (Ren and Govorsky, 2003).…”

Section: Histones Can Increase Transgene Expression In Several Transfmentioning

confidence: 99%

Overexpression of SeveralArabidopsisHistone Genes IncreasesAgrobacterium-Mediated Transformation and Transgene Expression in Plants

et al. 2009

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The Arabidopsis thaliana histone H2A-1 is important for Agrobacterium tumefaciens-mediated plant transformation. Mutation of HTA1, the gene encoding histone H2A-1, results in decreased T-DNA integration into the genome of Arabidopsis roots, whereas overexpression of HTA1 increases transformation frequency. To understand the mechanism by which HTA1 enhances transformation, we investigated the effects of overexpression of numerous Arabidopsis histones on transformation and transgene expression. Transgenic Arabidopsis containing cDNAs encoding histone H2A (HTA), histone H4 (HFO), and histone H3-11 (HTR11) displayed increased transformation susceptibility, whereas histone H2B (HTB) and most histone H3 (HTR) cDNAs did not increase transformation. A parallel increase in transient gene expression was observed when histone HTA, HFO, or HTR11 overexpression constructs were cotransfected with double-or single-stranded forms of a gusA gene into tobacco (Nicotiana tabacum) protoplasts. However, these cDNAs did not increase expression of a previously integrated transgene. We identified the N-terminal 39 amino acids of H2A-1 as sufficient to increase transient transgene expression in plants. After transfection, transgene DNA accumulates more rapidly in the presence of HTA1 than with a control construction. Our results suggest that certain histones enhance transgene expression, protect incoming transgene DNA during the initial stages of transformation, and subsequently increase the efficiency of Agrobacterium-mediated transformation. INTRODUCTIONAgrobacterium tumefaciens-mediated plant genetic transformation is an important experimental tool for investigation of various aspects of plant biology and for agricultural biotechnology. Development of this transformation technology represents the culmination of many decades of effort to improve tissue culture and plant genetic engineering techniques. Agrobacteriummediated transformation is based on a conjugative transfer-like process, which eventually takes the T-DNA to the host cell nucleus (Gelvin, 2000(Gelvin, , 2003a(Gelvin, , 2009Tzfira and Citovsky, 2003;Citovsky et al., 2007). After attachment of the bacterium to plant cells and induction of Agrobacterium virulence (vir) genes, a single-stranded DNA (the T-strand) is processed from the resident tumor-inducing or root-inducing plasmid and transported from the bacterium to the plant cell. In addition, a number of Virulence effector proteins, including VirD2 (attached to the T-strand), the single-strand DNA binding protein VirE2, plus VirE3, VirD5, and VirF are also transferred to the plant (Otten et al., 1984;Stahl et al., 1998;Vergunst et al., 2000Vergunst et al., , 2003Vergunst et al., , 2005Schrammeijer et al., 2003). These proteins are likely involved in protecting T-DNA from nuclease digestion, directing T-DNA to the nucleus, stripping proteins from the T-strand prior to integration, and integrating T-DNA into the plant genome. T-DNA integration occurs randomly into genomic DNA by the process of illegitimate recombination (...

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The Nonessential H2A N-Terminal Tail Can Function as an Essential Charge Patch on the H2A.Z Variant N-Terminal Tail

Cited by 33 publications

References 92 publications

Phosphorylation of the SQ H2A.X Motif Is Required for Proper Meiosis and Mitosis in Tetrahymena thermophila

Phosphorylation of the SQ H2A.X Motif Is Required for Proper Meiosis and Mitosis in Tetrahymena thermophila

Histone Variant H2A.Z and RNA Polymerase II Transcription Elongation

Overexpression of SeveralArabidopsisHistone Genes IncreasesAgrobacterium-Mediated Transformation and Transgene Expression in Plants

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