28 research outputs found

    Identification of Genome Targets of the DET1 Complex in Higher Plants

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    Light provides a major source of information from the environment during plant growth and development. Light-regulated gene expression is partly controlled by the phytochrome photoreceptors, which once activated, are imported into the nucleus where they bind and activate transcription factors such as PIF3. Coupled with this, the degradation of positively acting intermediates such as the transcription factor HY5 by COP1 and other ubiquitin ligases acts to repress photomorphogenesis in darkness. Another negative regulator of photomorphogenesis is DET1 (De-Etiolated 1), which forms part of a nuclear-localised complex with the plant homolog of UV- Damaged DNA Binding protein 1 (DDB1) and the E2 ubiquitin-ligase activating protein COP10 (Constitutive Photomorphogenic 10). Previously it was found that DET 1 binds chromatin via a direct interaction with the core histone H2B, suggesting that DET1 may repress light activated genes by interacting directly with their promoters. Here it is shown that DET1 forms part of a CUL4-based ubiquitin ligase complex and is localised to discrete foci throughout the nucleus. Chromatin Immunoprecipitation experiments show that in the dark DET 1 binds to the promoters of the light induced CAB2 and HEMA1 genes, and that this binding is abolished in the light, coincidental with the activation of these genes. DET 1 was also detected at the promoter of the light-repressed POR-A gene when it is in its repressed state as well as the promoter of the developmental^ regulated FT gene, again when this gene is repressed. Based on these data it is proposed that the DET1 complex binds the promoters of light- regulated genes in the dark and directly represses their transcription, either by chromatin remodelling and/or ubiquitin-mediated regulation of the transcriptional apparatus. Light causes the dissociation of the DET 1 complex from these promoters, allowing activation of these genes. DET1 may also play a more general role in genome maintenance during plant development

    DNA methylation variation in Arabidopsis has a genetic basis and shows evidence of local adaptation

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    Epigenome modulation in response to the environment potentially provides a mechanism for organisms to adapt, both within and between generations. However, neither the extent to which this occurs, nor the molecular mechanisms involved are known. Here we investigate DNA methylation variation in Swedish Arabidopsis thaliana accessions grown at two different temperatures. Environmental effects on DNA methylation were limited to transposons, where CHH methylation was found to increase with temperature. Genome-wide association mapping revealed that the extensive CHH methylation variation was strongly associated with genetic variants in both cis and trans, including a major trans-association close to the DNA methyltransferase CMT2. Unlike CHH methylation, CpG gene body methylation (GBM) on the coding region of genes was not affected by growth temperature, but was instead strongly correlated with the latitude of origin. Accessions from colder regions had higher levels of GBM for a significant fraction of the genome, and this was correlated with elevated transcription levels for the genes affected. Genome-wide association mapping revealed that this effect was largely due to trans-acting loci, a significant fraction of which showed evidence of local adaptation. These findings constitute the first direct link between DNA methylation and adaptation to the environment, and provide a basis for further dissecting how environmentally driven and genetically determined epigenetic variation interact and influence organismal fitness.Comment: 38 pages 4 figure

    Dynamics of a novel centromeric histone variant CenH3 reveals the evolutionary ancestral timing of centromere biogenesis

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    The centromeric histone H3 variant (CenH3) serves to target the kinetochore to the centromeres and thus ensures correct chromosome segregation during mitosis and meiosis. The Dictyostelium H3-like variant H3v1 was identified as the CenH3 ortholog. Dictyostelium CenH3 has an extended N-terminal domain with no similarity to any other known proteins and a histone fold domain at its C-terminus. Within the histone fold, α-helix 2 (α2) and an extended loop 1 (L1) have been shown to be required for targeting CenH3 to centromeres. Compared to other known and putative CenH3 histones, Dictyostelium CenH3 has a shorter L1, suggesting that the extension is not an obligatory feature. Through ChIP analysis and fluorescence microscopy of live and fixed cells, we provide here the first survey of centromere structure in amoebozoa. The six telocentric centromeres were found to mostly consist of all the DIRS-1 elements and to associate with H3K9me3. During interphase, the centromeres remain attached to the centrosome forming a single CenH3-containing cluster. Loading of Dictyostelium CenH3 onto centromeres occurs at the G2/prophase transition, in contrast to the anaphase/telophase loading of CenH3 observed in metazoans. This suggests that loading during G2/prophase is the ancestral eukaryotic mechanism and that anaphase/telophase loading of CenH3 has evolved more recently after the amoebozoa diverged from the animal linage

    Nuclear organisation and epigenetic regulation of gene expression in Dictyostelium discoideum

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    Supervisors: Prof. Dr. Wolfgang Nellen Prof. Dr. Markus Mania

    Transposons: a blessing curse

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    International audienc

    Advanced methylome analysis after bisulfite deep sequencing: an example in Arabidopsis.

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    Deep sequencing after bisulfite conversion (BS-Seq) is the method of choice to generate whole genome maps of cytosine methylation at single base-pair resolution. Its application to genomic DNA of Arabidopsis flower bud tissue resulted in the first complete methylome, determining a methylation rate of 6.7% in this tissue. BS-Seq reads were mapped onto an in silico converted reference genome, applying the so-called 3-letter genome method. Here, we present BiSS (Bisufite Sequencing Scorer), a new method applying Smith-Waterman alignment to map bisulfite-converted reads to a reference genome. In addition, we introduce a comprehensive adaptive error estimate that accounts for sequencing errors, erroneous bisulfite conversion and also wrongly mapped reads. The re-analysis of the Arabidopsis methylome data with BiSS mapped substantially more reads to the genome. As a result, it determines the methylation status of an extra 10% of cytosines and estimates the methylation rate to be 7.7%. We validated the results by individual traditional bisulfite sequencing for selected genomic regions. In addition to predicting the methylation status of each cytosine, BiSS also provides an estimate of the methylation degree at each genomic site. Thus, BiSS explores BS-Seq data more extensively and provides more information for downstream analysis

    Distribution of cytosine sequence context.

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    <p>(A) Frequency of sequence context in the reference genome. (B) Frequency of sequence context of methylated C according to BiSS and A3M.</p

    Global methylation level.

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    <p>Ratio of the number of mapped Cs divided by the number of mapped C plus T for all classified Cs. Black: Cs that are called methylated, White: C that are not called methylated.</p
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