Increased transcription in hydroxyurea-treated root meristem cells of Vicia faba

Winnicki, Konrad; Polit, Justyna Teresa; Maszewski, Janusz

doi:10.1007/s00709-012-0402-x

Cited by 8 publications

(5 citation statements)

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“…Our proteomic data further suggested that the chromatin association level of transcriptional machinery proteins including POLR2A and POLR2B was increased during G 1 and G 1 /S phases (Fig. 3B-C), which is characteristic of global transcriptional activation (53). In addition, cycline D1 expression regulates G 1 -S phase progression (54), and expression levels of this protein are modulated by the chromatin-modeling factor SNF2H and the CCAAT-binding protein C/EBP (55).…”

Section: Mass Spectrometric Identification and Quantification Of Thementioning

confidence: 91%

Profiling of the Chromatin-associated Proteome Identifies HP1BP3 as a Novel Regulator of Cell Cycle Progression

Dutta

Ren

Hao

et al. 2014

Molecular & Cellular Proteomics

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The chromatin-associated proteome (chromatome) regulates cellular gene expression by restricting access of transcriptional machinery to template DNA, and dynamic re-modeling of chromatin structure is required to regulate critical cell functions including growth and replication, DNA repair and recombination, and oncogenic transformation in progression to cancer. Central to the control of these processes is efficient regulation of the host cell cycle, which is maintained by rapid changes in chromatin conformation during normal cycle progression. A global overview of chromatin protein organization is therefore essential to fully understand cell cycle regulation, but the influence of the chromatome and chromatin binding topology on host cell cycle progression remains poorly defined. Here we used partial MNase digestion together with iTRAQ-based high-throughput quantitative proteomics to quantify chromatin-associated proteins during interphase progression. We identified a total of 481 proteins with high confidence that were involved in chromatin-dependent events including transcriptional regulation, chromatin reorganization, and DNA replication and repair, whereas the quantitative data revealed the temporal interactions of these proteins with chromatin during interphase progression. When combined with biochemical and functional assays, these data revealed a strikingly dynamic association of protein HP1BP3 with the chromatin complex during different stages of interphase, and uncovered a novel regulatory role for this molecule in transcriptional regulation. We report that HP1BP3 protein maintains heterochromatin integrity during G 1-S progression and regulates the duration of G 1 phase to critically influence cell proliferative capacity. Molecular & Cellular

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Section: Mass Spectrometric Identification and Quantification Of Thementioning

confidence: 91%

Profiling of the Chromatin-associated Proteome Identifies HP1BP3 as a Novel Regulator of Cell Cycle Progression

Dutta

Ren

Hao

et al. 2014

Molecular & Cellular Proteomics

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“…In a screen for resistance and sensitivity to acute hydroxyurea treatment, the authors identified mostly non-DDR genes as hits in their original analysis 11 , but not the accessory transcription factor gene TAF9. After Exorcise, analysis revealed TAF9 depletion as hypersensitising to acute hydroxyurea treatment, consistent with hydroxyurea's role in inducing transcriptional changes related to activation of the DDR [12][13][14] .…”

Section: Reannotation Of Published Ddr Crispr Screens Identifies Impr...mentioning

confidence: 78%

Genome-aware annotation of CRISPR guides validates targets in variant cell lines and enhances discovery in screens

Lam,

Thomas,

Jackson

2024

Preprint

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Pooled CRISPR-Cas9 genetic knockout screens are powerful high-throughput tools for identifying chemo-genetic, synthetic-lethal and synthetic-viability interactions and are used as a key step towards identifying disease-modifying knockout candidates and informing drug design and therapeutic regimens. CRISPR guide libraries are commercially available for purchase and have been widely applied in different cell lines. However, discrepancies between the genomes used to design CRISPR libraries and the genomes of the cells subjected to CRISPR screens lead to loss of signal or introduction of bias towards the most conserved genes. Here, we present an algorithm, EXOme-guided Reannotation of nuCleotIde SEquences (Exorcise), which uses sequence search and CRISPR target annotation to adapt existing CRISPR libraries to user-defined genomes and exomes. Applying Exorcise on 55 commercially available CRISPR-spCas9 knockout libraries for human and mouse, we found that all libraries have mis-annotations, and that design strategy affects off-target effects and targeting accuracy relative to a standard reference sequence. In simulations on synthetic data, we modelled common mis-annotations in CRISPR libraries and found that they adversely affected recovery of the ground truth for all genes except for those with the strongest signals. Finally, we reanalysed DepMap and DDRcs CRISPR screens with Exorcise annotations and found that strong hits were retained, and lower-confidence hits were strengthened. Use of Exorcise on DepMap with exomes inferred from transcriptomic expression data demonstrated that cell-line-aware reannotation is possible without whole-genome sequencing. Taken together, our results show that Exorcise is a powerful reannotation tool that focuses existing CRISPR libraries towards the cell line genome under investigation and allows post-hoc reanalysis of completed CRISPR screens. Exorcise is open-source software licenced under a Creative Commons Zero Universal 1.0 licence and is available at <https://github.com/SimonLammmm/exorcise>.

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“…Hartsuiker et al 2001 ). However, Winnicki et al ( 2013 ) revealed G1/S arrest in root meristems of V. faba treated for 24 h with 2.5 mM HU. Cytophotometric quantitative measurements of nuclear DNA after the 24-h replication block revealed two classes of cells: (i) those with 2-4C DNA content characterized by a low coefficient of chromatin condensation, and (ii) those with near 4C DNA content showing considerably greater condensation (comp.…”

Section: Discussionmentioning

confidence: 99%

“…After removing coverslips, the slides were dehydrated, air-dried, and embedded in Canada balsam (Merck, Germany). The nuclear DNA content was evaluated by means of microdensitometry using a Jenamed 2 microscope (Carl Zeiss, Jena, Germany) with the computer-aided Cytophotometer v1.2 (Forel, Lodz, Poland), as described in Winnicki et al ( 2013 ). The extinction of Feulgen-stained cell nuclei was measured at 565 nm and calibrated in arbitrary units, taking the values recorded for half-telophases and prophases from 24-h-negative control plants as reference standard of 2C [26.1 pg; (according to Rybaczek and Maszewski 2007a , and ref.…”

Section: Methodsmentioning

confidence: 99%

Ultrastructural changes associated with the induction of premature chromosome condensation in Vicia faba root meristem cells

Rybaczek

2014

Plant Cell Rep

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Key messagePCC induction is regulated by several signaling pathways, and all observed effects associated with PCC induction are strongly dependent on the mechanism of action of each PCC inducer used.AbstractElectron microscopic observations of cells with symptoms of premature chromosome condensation (PCC) showed that the interphase chromatin and mitotic chromosomes differed with respect to a chemical compound inducing PCC. Induction of this process under the influence of hydroxyurea and caffeine as well as hydroxyurea and sodium metavanadate led to a slight decrease in interphase chromatin condensation and the formation of chromosomes with a considerably loosened structure in comparison with the control. Incubation in the mixture of hydroxyurea and 2-aminopurine brought about clear chromatin dispersion in interphase and very strong mitotic chromosome condensation. Electron microscopic examinations also revealed the characteristic features of the structural organization of cytoplasm of Vicia faba root meristems, which seemed to be dependent on the type of the PCC inducer used. The presence of the following was observed: (i) large plastids filled with starch grains (caffeine), (ii) mitochondria and plastids of electron dense matrix with dilated invaginations of their internal membranes (2-aminopurine), and (iii) large mitochondria of electron clear matrix and plastids containing protein crystals in their interior (sodium metavanadate). Moreover, since caffeine causes either the most effective loosening of chromatin fibrils (within the prematurely condensed chromosomes) or induction of starch formation (in the plastids surrounding the nuclei), this may be a proof that demonstrates the existence of a link between physical accessibility to chromatin and the effectiveness of cellular signaling (e.g., phosphothreonine-connected).

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Increased transcription in hydroxyurea-treated root meristem cells of Vicia faba

Cited by 8 publications

References 50 publications

Profiling of the Chromatin-associated Proteome Identifies HP1BP3 as a Novel Regulator of Cell Cycle Progression

Profiling of the Chromatin-associated Proteome Identifies HP1BP3 as a Novel Regulator of Cell Cycle Progression

Genome-aware annotation of CRISPR guides validates targets in variant cell lines and enhances discovery in screens

Ultrastructural changes associated with the induction of premature chromosome condensation in Vicia faba root meristem cells

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