15 research outputs found

    Multiple histone modifications in euchromatin promote heterochromatin formation by redundant mechanisms in Saccharomyces cerevisiae

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    <p>Abstract</p> <p>Background</p> <p>Methylation of lysine 79 on histone H3 by Dot1 is required for maintenance of heterochromatin structure in yeast and humans. However, this histone modification occurs predominantly in euchromatin. Thus, Dot1 affects silencing by indirect mechanisms and does not act by the recruitment model commonly proposed for histone modifications. To better understand the role of H3K79 methylation gene silencing, we investigated the silencing function of Dot1 by genetic suppressor and enhancer analysis and examined the relationship between Dot1 and other global euchromatic histone modifiers.</p> <p>Result</p> <p>We determined that loss of H3K79 methylation results in a partial silencing defect that could be bypassed by conditions that promote targeting of Sir proteins to heterochromatin. Furthermore, the silencing defect in strains lacking Dot1 was dependent on methylation of H3K4 by Set1 and histone acetylation by Gcn5, Elp3, and Sas2 in euchromatin. Our study shows that multiple histone modifications associated with euchromatin positively modulate the function of heterochromatin by distinct mechanisms. Genetic interactions between Set1 and Set2 suggested that the H3K36 methyltransferase Set2, unlike most other euchromatic modifiers, negatively affects gene silencing.</p> <p>Conclusion</p> <p>Our genetic dissection of Dot1's role in silencing in budding yeast showed that heterochromatin formation is modulated by multiple euchromatic histone modifiers that act by non-overlapping mechanisms. We discuss how euchromatic histone modifiers can make negative as well as positive contributions to gene silencing by competing with heterochromatin proteins within heterochromatin, within euchromatin, and at the boundary between euchromatin and heterochromatin.</p

    Dot1 binding induces chromatin rearrangements by histone methylation-dependent and -independent mechanisms

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    <p>Abstract</p> <p>Background</p> <p>Methylation of histone H3 lysine 79 (H3K79) by Dot1 is highly conserved among species and has been associated with both gene repression and activation. To eliminate indirect effects and examine the direct consequences of Dot1 binding and H3K79 methylation, we investigated the effects of targeting Dot1 to different positions in the yeast genome.</p> <p>Results</p> <p>Targeting Dot1 did not activate transcription at a euchromatic locus. However, chromatin-bound Dot1 derepressed heterochromatin-mediated gene silencing over a considerable distance. Unexpectedly, Dot1-mediated derepression was established by both a H3K79 methylation-dependent and a methylation-independent mechanism; the latter required the histone acetyltransferase Gcn5. By monitoring the localization of a fluorescently tagged telomere in living cells, we found that the targeting of Dot1, but not its methylation activity, led to the release of a telomere from the repressive environment at the nuclear periphery. This probably contributes to the activity-independent derepression effect of Dot1.</p> <p>Conclusions</p> <p>Targeting of Dot1 promoted gene expression by antagonizing gene repression through both histone methylation and chromatin relocalization. Our findings show that binding of Dot1 to chromatin can positively affect local gene expression by chromatin rearrangements over a considerable distance.</p

    Shugoshin biases chromosomes for biorientation through condensin recruitment to the pericentromere

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    To protect against aneuploidy, chromosomes must attach to microtubules from opposite poles (‘biorientation’) prior to their segregation during mitosis. Biorientation relies on the correction of erroneous attachments by the aurora B kinase, which destabilizes kinetochore-microtubule attachments that lack tension. Incorrect attachments are also avoided because sister kinetochores are intrinsically biased towards capture by microtubules from opposite poles. Here, we show that shugoshin acts as a pericentromeric adaptor that plays dual roles in biorientation in budding yeast. Shugoshin maintains the aurora B kinase at kinetochores that lack tension, thereby engaging the error correction machinery. Shugoshin also recruits the chromosome-organizing complex, condensin, to the pericentromere. Pericentromeric condensin biases sister kinetochores towards capture by microtubules from opposite poles. Our findings uncover the molecular basis of the bias to sister kinetochore capture and expose shugoshin as a pericentromeric hub controlling chromosome biorientation. DOI: http://dx.doi.org/10.7554/eLife.01374.00

    A Barcode Screen for Epigenetic Regulators Reveals a Role for the NuB4/HAT-B Histone Acetyltransferase Complex in Histone Turnover

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    Dynamic modification of histone proteins plays a key role in regulating gene expression. However, histones themselves can also be dynamic, which potentially affects the stability of histone modifications. To determine the molecular mechanisms of histone turnover, we developed a parallel screening method for epigenetic regulators by analyzing chromatin states on DNA barcodes. Histone turnover was quantified by employing a genetic pulse-chase technique called RITE, which was combined with chromatin immunoprecipitation and high-throughput sequencing. In this screen, the NuB4/HAT-B complex, containing the conserved type B histone acetyltransferase Hat1, was found to promote histone turnover. Unexpectedly, the three members of this complex could be functionally separated from each other as well as from the known interacting factor and histone chaperone Asf1. Thus, systematic and direct interrogation of chromatin structure on DNA barcodes can lead to the discovery of genes and pathways involved in chromatin modification and dynamics

    Recombination-Induced tag exchange (RITE) cassette series to monitor protein dynamics in Saccharomyces cerevisiae

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    This is an open-access article distributed under the terms of the Creative Commons Attribution Unported License.Proteins are not static entities. They are highly mobile and their steady state levels are achieved by a balance between ongoing synthesis and degradation. The dynamic properties of a protein can have important consequences for its function. For example, when a protein is degraded and replaced by a newly synthesized one, post-translational modifications are lost and need to be reincorporated in the new molecules. Protein stability and mobility are also relevant for duplication of macromolecular structures or organelles, which involves coordination of protein inheritance with the synthesis and assembly of newly synthesized proteins. To measure protein dynamics we recently developed a genetic pulse-chase assay called Recombination-Induced Tag Exchange (RITE). RITE has been successfully used in Saccharomyces cerevisiae to measure turnover and inheritance of histone proteins, to study changes in post-translational modifications on aging proteins, and to visualize the spatiotemporal inheritance of protein complexes and organelles in dividing cells. Here we describe a series of successful RITE cassettes that are designed for biochemical analyses, genomics studies, as well as single cell fluorescence applications. Importantly, the genetic nature and the stability of the tag-switch offer the unique possibility to combine RITE with high-throughput screening for protein dynamics mutants and mechanisms. The RITE cassettes are widely applicable, modular by design, and can therefore be easily adapted for use in other cell types or organisms.This project was sponsored by the Netherlands Genomics Initiative and by The Netherlands Organization for Scientific Research.Peer Reviewe

    Decoding the chromatin proteome of a single genomic locus by DNA sequencing

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    <div><p>Transcription, replication, and repair involve interactions of specific genomic loci with many different proteins. How these interactions are orchestrated at any given location and under changing cellular conditions is largely unknown because systematically measuring protein–DNA interactions at a specific locus in the genome is challenging. To address this problem, we developed Epi-Decoder, a Tag-chromatin immunoprecipitation-Barcode-Sequencing (TAG-ChIP-Barcode-Seq) technology in budding yeast. Epi-Decoder is orthogonal to proteomics approaches because it does not rely on mass spectrometry (MS) but instead takes advantage of DNA sequencing. Analysis of the proteome of a transcribed locus proximal to an origin of replication revealed more than 400 interacting proteins. Moreover, replication stress induced changes in local chromatin proteome composition prior to local origin firing, affecting replication proteins as well as transcription proteins. Finally, we show that native genomic loci can be decoded by efficient construction of barcode libraries assisted by clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9). Thus, Epi-Decoder is an effective strategy to identify and quantify in an unbiased and systematic manner the proteome of an individual genomic locus by DNA sequencing.</p></div

    Chromatin rewiring upon HU treatment.

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    <p>(A) DNA regions replicated in the HU arrest were determined by DNA copy number assessment. The DNA coverage in bins of 200 bp is plotted across the genome for untreated and HU-treated samples. The lower panel shows a zoom-in of chromosome IV, which contains the barcoded locus on the left arm (indicated by the arrow). Replication timing in the absence of HU was obtained from Alvino and colleagues [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005542#pbio.2005542.ref036" target="_blank">36</a>]. The percentage of replicated DNA is plotted for each bin at 10, 12.5, 15, 17.5, 25, and 40 minutes after release from a G1 arrest, as indicated by the shades of grey. (B) RT-qPCR shows relative mRNA expression of <i>KanMX/HphMX</i> in untreated and HU-treated samples. The untreated samples were set to 1. The average of 3 biological replicates is shown; error bars indicate SD. (C) Volcano plots showing the ratio of binding in HU-treated/untreated for factors that were significantly enriched in either one or both of the conditions. The coloured dots are factors with significantly different binding scores (FDR < 0.05). BC_DN, downstream barcode; BC_UP, upstream barcode; FDR, false discovery rate; HU, hydroxyurea; ORC, origin recognition complex; RT-qPCR, quantitative reverse transcription PCR.</p

    Capturing the chromatin interactome with Epi-Decoder.

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    <p>(A) Scatter plot of ChIP/input values, which can be interpreted as binding scores, of BC_UP versus BC_DN. For some factors, only the BC_UP or BC_DN value was available. In these cases, the missing value was set to −1 in order to still visualize the remaining value. Factors are coloured based on known functions or complexes. Triangles represent histone proteins. The average values of 6 replicates are shown. (B) Enrichment plot for 3 categories: initiation (TFIID-E and GRFs), termination (Cleavage and Polyadenylation and THO complex), and replication (ORC and MCM) factors. Binders were ranked based on the BC_UP/BC_DN ratio. The top part shows the running sum enrichment for each category. Initiation factors were significantly enriched at BC_UP (<i>p</i> = 3.18<sup>−3</sup>) and termination and replication at BC_DN (<i>p</i> = 2.03<sup>−7</sup> and <i>p</i> = 1.06<sup>−5</sup>). The bottom lines indicate the factors represented in the categories. (C) Illustration of the different protein complexes that Epi-Decoder identified at the barcoded <i>KanMX</i> gene at the <i>HO</i> locus. BC_DN, downstream barcode; BC_UP, upstream barcode; ChIP, chromatin immunoprecipitation; GRF, general regulatory factor; IP, immunoprecipitation; MCM, minichromosome maintenance; ORC, origin recognition complex.</p
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