1,721,084 research outputs found
Epigenomic consequences of immortalized plant cell suspension culture
Get PDFPlant cells grown in culture exhibit genetic and epigenetic instability. Using a combination of chromatin immunoprecipitation and DNA methylation profiling on tiling microarrays, we have mapped the location and abundance of histone and DNA modifications in a continuously proliferating, dedifferentiated cell suspension culture of Arabidopsis. We have found that euchromatin becomes hypermethylated in culture and that a small percentage of the hypermethylated genes become associated with heterochromatic marks. In contrast, the heterochromatin undergoes dramatic and very precise DNA hypomethylation with transcriptional activation of specific transposable elements (TEs) in culture. High throughput sequencing of small interfering RNA (siRNA) revealed that TEs activated in culture have increased levels of 21-nucleotide (nt) siRNA, sometimes at the expense of the 24-nt siRNA class. In contrast, TEs that remain silent, which match the predominant 24-nt siRNA class, do not change significantly in their siRNA profiles. These results implicate RNA interference and chromatin modification in epigenetic restructuring of the genome following the activation of TEs in immortalized cell culture
Psip1/Ledgf p52 binds methylated histone H3K36 and splicing factors and contributes to the regulation of alternative splicing
Get PDFIncreasing evidence suggests that chromatin modifications have important roles in modulating constitutive or alternative splicing. Here we demonstrate that the PWWP domain of the chromatin-associated protein Psip1/Ledgf can specifically recognize tri-methylated H3K36 and that, like this histone modification, the Psip1 short (p52) isoform is enriched at active genes. We show that the p52, but not the long (p75), isoform of Psip1 co-localizes and interacts with Srsf1 and other proteins involved in mRNA processing. The level of H3K36me3 associated Srsf1 is reduced in Psip1 mutant cells and alternative splicing of specific genes is affected. Moreover, we show altered Srsf1 distribution around the alternatively spliced exons of these genes in Psip1 null cells. We propose that Psip1/p52, through its binding to both chromatin and splicing factors, might act to modulate splicing
DNMT3L modulates significant and distinct flanking sequence preference for DNA methylation by DNMT3A and DNMT3B in vivo.
Get PDFThe DNTM3A and DNMT3B de novo DNA methyltransferases (DNMTs) are responsible for setting genomic DNA methylation patterns, a key layer of epigenetic information. Here, using an in vivo episomal methylation assay and extensive bisulfite methylation sequencing, we show that human DNMT3A and DNMT3B possess significant and distinct flanking sequence preferences for target CpG sites. Selection for high or low efficiency sites is mediated by the base composition at the -2 and +2 positions flanking the CpG site for DNMT3A, and at the -1 and +1 positions for DNMT3B. This intrinsic preference reproducibly leads to the formation of specific de novo methylation patterns characterized by up to 34-fold variations in the efficiency of DNA methylation at individual sites. Furthermore, analysis of the distribution of signature methylation hotspot and coldspot motifs suggests that DNMT flanking sequence preference has contributed to shaping the composition of CpG islands in the human genome. Our results also show that the DNMT3L stimulatory factor modulates the formation of de novo methylation patterns in two ways. First, DNMT3L selectively focuses the DNA methylation machinery on properly chromatinized DNA templates. Second, DNMT3L attenuates the impact of the intrinsic DNMT flanking sequence preference by providing a much greater boost to the methylation of poorly methylated sites, thus promoting the formation of broader and more uniform methylation patterns. This study offers insights into the manner by which DNA methylation patterns are deposited and reveals a new level of interplay between members of the de novo DNMT family
A function of thymine DNA glycosylase-initiated DNA repair in maintaining epigenome stability
Get PDFThe Thymine DNA Glycosylase (TDG) was initially discovered by its ability to excise the deamination
products of cytosine and 5-methylcytosine (5-mC), and therefore thought to initiate base excision
repair (BER) of the resulting G•U and G•T mismatches. Later, TDG was also found to act in concert
with transcription factors in the regulation of gene expression.
With its apparently two-sided nature, TDG has riddled researchers for many years and the stimuli
and interactions that control TDG function are still under investigation. The aim of my thesis was to
dissect the role of TDG in DNA repair with a focus on its regulation by post-translational modification,
and to investigate how TDG-initiated BER contributes to epigenetic stability at CpG islands (CGIs)
during cell differentiation.
Both described functions of TDG, in DNA repair and in the regulation of gene expression, require its
post-translational modification and non-covalent interaction with the small ubiquitin-like modifiers,
SUMO1 and SUMO2/3. Extensive biochemical studies by our laboratory have shown that
SUMOylation of TDG may induce its dissociation from the abasic (AP-) site after base excision.
However, in vivo evidence corroborating an involvement of SUMOylation in TDG-dependent BER has
been pending and the function of non-covalent SUMO-binding has remained elusive. I thus
generated a Fluorescence Resonance Energy Transfer (FRET) system to monitor the interaction
between TDG and SUMO1 or SUMO3 in cells. I was able to confirm a modulation of the SUMO1-TDG
interaction dynamics in response to DNA damage, whereas the interaction with SUMO3 remained
unaffected, suggesting that modification by SUMO3 might regulate TDG function in a context other
than DNA repair.
To investigate the biological function of TDG genetically, we generated a Tdg knockout mouse. In
contrast to any other known DNA glycosylase, deletion of Tdg caused embryonic lethality. Further
characterization of MEFs isolated from TDG-proficient and -deficient embryos revealed no evidence
for a DNA repair defect, but a significant number of misregulated genes in differentiated Tdg-/- cells,
as well as a loss of active histone marks, gain of repressive histone modifications and an
accumulation of 5-mC at CGI promoters. A phenotype we did not observe in embryonic stem cells.
From these data, we proposed a dual function of TDG in maintaining active chromatin states at
promoters in differentiating cells, first by structurally coordinating histone modifying enzymes and
second by counteracting errors of the DNA methylation machinery by initiating repair of aberrantly
methylated cytosines in CGIs. Consistent with a TDG-dependent engagement of DNA repair at such
sites, we found BER factors to associate with these promoters and DNA repair intermediates to
accumulate in differentiating cells in a TDG dependent manner.
To investigate further how TDG is involved in DNA methylation control, we mapped DNA methylation
in the genomes of TDG-proficient and -deficient mouse embryonic stem cells (ESCs), neuronal
progenitor cells (NPs) and MEFs and found differential methylation to arise only with differentiation.
Further characterization of the resulting differentially methylated regions (DMRs) revealed that those
overlapping with a CGI were almost exclusively hypomethylated in TDG-deficient compared to -proficient cells,
reflecting a failure to establish methylation at these CGIs during differentiation. In
search of the reason for this failure in a 24 h differentiation timecourse, we found global 5-mC levels
to rise with differentiation in cells lacking TDG activity, in parallel to the generation of the final
products of TET-protein catalyzed 5-mC oxidation, 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-
caC), the latter two of which are proposed intermediates of active DNA demethylation and
substrates for TDG. Differentiation thus appeared to induce methylation but also the intermediates
of active demethylation. We therefore analyzed 5-mC and 5-caC levels at the CGI DMRs and found
both to rise with differentiation in wildtype cells, suggesting that the loss of pluripotency induces a
cycle of DNA methylation and demethylation specific CGIs. In Tdg knockout cells, though, this
induction appeared to fail whereas in cells expressing a catalytically dead mutant TDG (TDG-cat), the
cycle of methylation and demethylation was induced but blocked by the inability of TDG-cat to excise
5-caC.
Taken together, in collaboration with colleagues from different laboratories I was able to show that
differentiation triggers a state of high epigenetic plasticity at these CGIs and that catalytically active
TDG is required to maintain an equilibrium of DNA methylation and demethylation. The imbalance of
epigenetic marks resulting from knockout of TDG disrupts gene expression programs and the
accumulation of aberrations eventually leads to loss of viability on the cellular and on the organismic
level
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Non-canonical roles of mammalian heterochromatin protein 1 (HP1) homologs
Get PDFThe human body is composed of hundreds of different tissues, all containing same genetic information, yet defined by unique gene expression patterns. In order to achieve this, multicellular organisms evolved regulatory mechanisms that go beyond the mere DNA sequence. Unlike in prokaryotes, where DNA is freely accessible to the transcriptional machinery, DNA in eukaryotic cells is wrapped around histone proteins, forming a structure called chromatin. Importantly, chromatin can be regulated by various post-translational modifications of the histone proteins. Such modifications are generally assumed to directly affect the compaction of chromatin and/or act as a recruitment platform for chromatin factors that relax (activate) or condense (repress) chromatin. One of the repressive histone marks, histone 3 lysine 9 methylation (H3K9me), is recognised by members of the HP1 family and/or by other proteins that have the unique ability to spread along chromatin, compacting it, and ultimately forming large inaccessible domains referred to as heterochromatin. Over the past decade, however, the view of HP1s as rigid silencers has been gradually challenged, as it was found that heterochromatic regions produce RNA, and that HP1s are highly mobile molecules. In addition, HP1 proteins were shown to associate with RNAs, and to also associate with chromatin lacking the H3K9me mark. These findings raised several fundamental questions: Is HP1 activity regulated by RNA? How are HP1 proteins recruited to sites lacking H3K9me, and what is their role at those sites?
One aim of my PhD project was to elucidate potential roles of RNA in modulating HP1 activity. For this, I used biochemical methods to dissect RNA binding properties of mammalian HP1 proteins in vitro. My work revealed that one of the HP1 homologs, HP1 alpha, interacts with RNA when bound to H3K9me-marked nucleosomes. The physiological role of such interaction could be stabilisation of binding to heterochromatin, or alternatively, eviction from heterochromatin. The major goal of my PhD project, however, was to investigate the mechanism of HP1 recruitment to chromatin lacking the H3K9me mark. To do so, I was using mouse embryonic stem cells (mESCs) as a model system. I have exploited recent advances in genome editing/CRISPR-Cas9 to delete or endogenously tag individual HP1 homologs or various combinations thereof. Genome- and proteome-wide studies subsequently revealed a novel protein complex, which I dubbed “ChAHP”. ChAHP contains two HP1 homologs (HP1 beta and/or HP1 gamma), the transcription factor Adnp, and the chromatin remodeller Chd4. In collaboration with the group of Nicolas Thomä, we have reconstituted the ChAHP complex in vitro from insect cells and dissected the individual interactions. Together with my proteomics experiments in mESCs, this revealed Adnp as a core bridging module interacting with Chd4 and HP1s. Using ChIP-sequencing, we identified over 15 000 ChAHP-bound genomic sites. Importantly, these sites are devoid of H3K9me2 or H3K9me3. Instead, the complex is targeted via a highly conserved DNA motif recognized by Adnp, and deletion of Adnp or of the DNA motif depletes HP1 binding at the ChAHP sites. In addition, deletion of Adnp or HP1s leads to derepression of lineage-specifying genes bound by ChAHP. However, unlike in case of canonical HP1 silencing, which involves H3K9me and results in formation of a broad heterochromatic domain, ChAHP silencing occurs locally by restricting access to its sites. I propose that this prevents other regulators, including transcriptional activators, from accessing the corresponding DNA sites. Finally, my results provide first insights into the molecular mechanism of a disease that is associated with mutations in the ADNP gene, Helsmoortel-Van der Aa syndrome. Mutant Adnp found in Helsmoortel-Van der Aa patients fails to interact with HP1 proteins, and therefore cannot target HP1s to the chromatin. In summary, my work revealed that HP1 proteins can be recruited to genomic loci in a DNA sequence-specific, H3K9 methylation-independent, manner via an interaction with the transcription factor Adnp, and I demonstrated that H3K9 methylation, unlike in canonical silencing, is not required for repression of ChAHP target genes
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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