CRISPR-Cas gene editing holds substantial promise in many biomedical disciplines and basic research. Due to the important functional implications of non-histone chromosomal protein HMG-14 (HMGN1) in regulating chromatin structure and tumor immunity, gene knockout of HMGN1 is performed by CRISPR in cancer cells and the following proteomic regulation events are studied. In particular, DIA mass spectrometry (DIA-MS) is utilized, and more than 6200 proteins (protein- FDR 1%) and more than 82 000 peptide precursors are reproducibly measured in the single MS shots of 2 h. HMGN1 protein deletion is confidently verified by DIA-MS in all of the clone- and dish- replicates following CRISPR. Statistical analysis reveals 147 proteins change their expressions significantly after HMGN1 knockout. Functional annotation and enrichment analysis indicate the deletion of HMGN1 induces histone inactivation, various stress pathways, remodeling of extracellular proteomes, cell proliferation, as well as immune regulation processes such as complement and coagulation cascade and interferon alpha/ gamma response in cancer cells. These results shed new lights on the cellular functions of HMGN1. It is suggested that DIA-MS can be reliably used as a rapid, robust, and cost-effective proteomic-screening tool to assess the outcome of the CRISPR experiments.
- MeSH
- chromatin fyziologie MeSH
- CRISPR-Cas systémy MeSH
- delece genu * MeSH
- editace genu metody MeSH
- HeLa buňky MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- proliferace buněk genetika MeSH
- protein HMGN1 genetika MeSH
- proteomika metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Microbial-associated molecular patterns activate several MAP kinases, which are major regulators of the innate immune response in Arabidopsis thaliana that induce large-scale changes in gene expression. Here, we determine whether microbial-associated molecular pattern-triggered gene expression involves modifications at the chromatin level. RESULTS: Histone acetylation and deacetylation are major regulators of microbial-associated molecular pattern-triggered gene expression and implicate the histone deacetylase HD2B in the reprogramming of defence gene expression and innate immunity. The MAP kinase MPK3 directly interacts with and phosphorylates HD2B, thereby regulating the intra-nuclear compartmentalization and function of the histone deacetylase. CONCLUSIONS: By studying a number of gene loci that undergo microbial-associated molecular pattern-dependent activation or repression, our data reveal a mechanistic model for how protein kinase signaling directly impacts chromatin reprogramming in plant defense.
- MeSH
- Arabidopsis imunologie MeSH
- chromatin fyziologie MeSH
- flagelin imunologie MeSH
- fosforylace MeSH
- fyziologický stres MeSH
- histondeacetylasy metabolismus MeSH
- histony metabolismus MeSH
- imunita rostlin * MeSH
- mitogenem aktivované proteinkinasy kinas metabolismus MeSH
- přirozená imunita MeSH
- proteiny huseníčku metabolismus MeSH
- restrukturace chromatinu * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Developmental exposure to environmental factors has been linked to obesity risk later in life. Nuclear receptors are molecular sensors that play critical roles during development and, as such, are prime candidates to explain the developmental programming of disease risk by environmental chemicals. We have previously characterized the obesogen tributyltin (TBT), which activates the nuclear receptors peroxisome proliferator-activated receptor γ (PPARγ) and retinoid X receptor (RXR) to increase adiposity in mice exposed in utero. Mesenchymal stem cells (MSCs) from these mice are biased toward the adipose lineage at the expense of the osteoblast lineage, and MSCs exposed to TBT in vitro are shunted toward the adipose fate in a PPARγ-dependent fashion. To address where in the adipogenic cascade TBT acts, we developed an in vitro commitment assay that permitted us to distinguish early commitment to the adipose lineage from subsequent differentiation. TBT and RXR activators (rexinoids) had potent effects in committing MSCs to the adipose lineage, whereas the strong PPARγ activator rosiglitazone was inactive. We show that activation of RXR is sufficient for adipogenic commitment and that rexinoids act through RXR to alter the transcriptome in a manner favoring adipogenic commitment. RXR activation alters expression of enhancer of zeste homolog 2 (EZH2) and modifies genome-wide histone 3 lysine 27 trimethylation (H3K27me3) in promoting adipose commitment and programming subsequent differentiation. These data offer insights into the roles of RXR and EZH2 in MSC lineage specification and shed light on how endocrine-disrupting chemicals such as TBT can reprogram stem cell fate.
- MeSH
- adipogeneze účinky léků genetika fyziologie MeSH
- buněčná diferenciace účinky léků genetika MeSH
- chromatin účinky léků fyziologie MeSH
- endokrinní disruptory farmakologie MeSH
- epigeneze genetická účinky léků MeSH
- exprese genu účinky léků MeSH
- EZH2 protein genetika MeSH
- genový knockdown veterinární MeSH
- mezenchymální kmenové buňky cytologie MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- obezita etiologie MeSH
- PPAR gama fyziologie MeSH
- retinoidní X receptory účinky léků fyziologie MeSH
- sekvenční analýza RNA veterinární MeSH
- trialkylcínové sloučeniny farmakologie MeSH
- tukové buňky cytologie MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
Epigenetické změny jsou úzce spjaty s rozvojem a progresí nádorového onemocnění a lze je korigovat aplikací epigenetických cytostatik. Cílem tohoto sdělení je podat přehled o molekulárních mechanizmech epigenetických cytostatik a o jejich možném klinickém použití. Většina těchto chemoterapeutik je předmětem klinických studií. Jen 2 demetylující látky, t.j. inhibitory DNA metyltransferáz (5-azacytidin a decitabin) jsou schváleny k léčbě myelodysplastického syndromu a několik cytostatik ze skupiny inhibitorů histondeacetyláz (vorinostat, romidepsin a panobinostat) lze použít v léčbě hematologických malignit, zejména k léčbě refrakterních a relabujích kožních T lymfomů. Klíčová slova: epigenetická cytostatika – inhibitory DNA metyltransferáz – inhibitory histondeacetyláz
Epigenetic modification have been causally linked to cancer development and progression, and are potentially reversible by treatments with epigenetic cancer drugs. The aim of this review is to give an overview of the basic current knowledge on molecular mechanisms of epigenetic cancer drugs and their possible clinical use. Many of them are in inclinical trials. However only two demethylating agents ie. inhibitors of DNA methyltransferase (5-azacytidin and decitabin) are approved in the treatment of myelodysplastic syndrome and a few inhibitors of histonacetylase (vorinostat, romidepsin and panobinostat) are approved in the treatment of hematological malignancies, particularly in refractory or relapsed cutaneous T‑cell lymhoma. Key words: epigenetic cancer drugs – inhibitors of DNA methyltransferase – inhibitors of histondeacetylase
- Klíčová slova
- modifikace histonů, inhibitory histon deacetyláz, inhibitory DNA metyltransferáz,
- MeSH
- azacytidin MeSH
- chromatin * fyziologie genetika MeSH
- cytostatické látky * MeSH
- epigeneze genetická MeSH
- exprese genu účinky léků MeSH
- fyziologie buňky MeSH
- histonacetyltransferasy * antagonisté a inhibitory MeSH
- histondeacetylasy MeSH
- histony * účinky léků MeSH
- lidé MeSH
- metylace DNA * MeSH
- nádorová transformace buněk MeSH
- nádory farmakoterapie MeSH
- Check Tag
- lidé MeSH
During meiosis, pairing of homologous chromosomes and their synapsis are essential prerequisites for normal male gametogenesis. Even limited autosomal asynapsis often leads to spermatogenic impairment, the mechanism of which is not fully understood. The present study was aimed at deliberately increasing the size of partial autosomal asynapsis and analysis of its impact on male meiosis. For this purpose, we studied the effect of t(12) haplotype encompassing four inversions on chromosome 17 on mouse autosomal translocation T(16;17)43H (abbreviated T43H). The T43H/T43H homozygotes were fully fertile in both sexes, while +/T43H heterozygous males, but not females, were sterile with meiotic arrest at late pachynema. Inclusion of the t(12) haplotype in trans to the T43H translocation resulted in enhanced asynapsis of the translocated autosome, ectopic phosphorylation of histone H2AX, persistence of RAD51 foci, and increased gene silencing around the translocation break. Increase was also on colocalization of unsynapsed chromatin with sex body. Remarkably, we found that transcriptional silencing of the unsynapsed autosomal chromatin precedes silencing of sex chromosomes. Based on the present knowledge, we conclude that interference of meiotic silencing of unsynapsed autosomes with meiotic sex chromosome inactivation is the most likely cause of asynapsis-related male sterility.
- MeSH
- biologické modely MeSH
- chromatin metabolismus fyziologie MeSH
- chromozomy genetika metabolismus MeSH
- genetická transkripce genetika MeSH
- hybridizace in situ fluorescenční MeSH
- meióza genetika fyziologie MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- párování chromozomů genetika MeSH
- pohlavní chromozomy genetika metabolismus MeSH
- regulace genové exprese MeSH
- translokace genetická genetika MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The multiprotein complexes known as condensins (I and II) are major players in chromosome dynamics in mitotic and meiotic cells. Here, we report for the first time the detection of different condensin subunits from both complexes in mammalian oocytes. Using immunoblotting analysis we examined expression levels of condensin subunits during meiotic maturation of porcine oocytes. The expression of the core subunit structural maintenance of chromosomes 2 (SMC2), identical in both condensin complexes, did not change significantly during maturation. Similarly, there was no significant change in the expression of the chromosome associated protein (CAP)-H and CAP-H2 subunits, components of condensin I and II, respectively. Conversely, the expression profiles of CAP-G, CAP-D2 (condensin I) and CAP-D3 (condensin II) were more interesting. At least two isoforms of the CAP-D2 subunit were detected, along with three isoforms of the CAP-D3 and CAP-G subunits. We suggest that this diverse migration of subunit isoforms is due to post-translational modification. Earlier, it was reported that non-SMC proteins are phosphorylated by cyclin-dependent kinase 1. In the present study, we analysed the phosphorylation status of the three subunits in oocyte extracts using alkaline phosphatase treatment and we found that at least the fastest migrating form of CAP-D3 was likely to be phosphorylated in maturing porcine oocytes. In addition, the localisation of CAP-H and CAP-H2 subunits was examined using immunofluorescence staining with specific antibodies, as well as following microinjection of their enhanced green fluorescent protein-tagged mRNA into germinal vesicle-stage oocytes. CAP-H was found in the cytoplasm, whereas CAP-H2 was localised within the nucleus.
- MeSH
- adenosintrifosfatasy metabolismus fyziologie ultrastruktura MeSH
- chromatin fyziologie MeSH
- chromozomy fyziologie MeSH
- DNA vazebné proteiny metabolismus fyziologie ultrastruktura MeSH
- fluorescenční mikroskopie veterinární MeSH
- imunoblotting veterinární MeSH
- konfokální mikroskopie veterinární MeSH
- meióza fyziologie MeSH
- multiproteinové komplexy metabolismus fyziologie ultrastruktura MeSH
- oocyty fyziologie MeSH
- podjednotky proteinů MeSH
- posttranslační úpravy proteinů MeSH
- prasata fyziologie MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Here, we would like to point out important milestones in the study of nuclear radial positioning and gene expression during differentiation processes. In addition, changes in the histone signature that significantly precede various differentiation pathways are reviewed. We address the regulatory functions of chromatin structure and histone epigenetic marks that give rise to gene expression patterns that are specific to distinct differentiation pathways. The functional relevance of nuclear architecture and epigenetic traits is preferentially discussed in the context of in vitro induced enterocytic differentiation and pluripotent or differentiated embryonic stem cells. We especially focus on the recapitulation of nuclear events that have been characterized for some genes and proto-oncogenes that are important for development and differentiation.
- MeSH
- buněčná diferenciace MeSH
- chromatin chemie fyziologie MeSH
- embryonální kmenové buňky cytologie MeSH
- histony genetika metabolismus MeSH
- lidé MeSH
- pluripotentní kmenové buňky cytologie MeSH
- regulace genové exprese MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
According to a general paradigm, proper DNA duplication from each replication origin is ensured by two protein complexes termed replisomes. In prokaryotes and in budding yeast Saccharomyces cerevisiae, these two replisomes seem to be associated with one another until DNA replication initiated from the origin has finished. This arrangement results in the formation of the loop of newly synthesized DNA. However, arrangement of replisomes in other eukaryotic organisms including vertebrate cells is largely unknown. Here, we used in vivo labeling of DNA segments in combination with the electron microscopy tomography to describe the organization of replisomes in human HeLa cells. The experiments were devised in order to distinguish between a model of independent replisomes and a model of replisome couples. The comparative analysis of short segments of replicons labeled in pulse-chase experiments of various length shows that replisomes in HeLa cells are organized into the couples during DNA replication. Moreover, our data enabled to suggest a new model of the organization of replicated DNA. According to this model, replisome couples produce loop with the associated arms in the form of four tightly associated 30nm fibers.
- MeSH
- bromodeoxyuridin metabolismus MeSH
- buněčné jádro metabolismus ultrastruktura MeSH
- chromatin fyziologie ultrastruktura MeSH
- deoxyuracilnukleotidy metabolismus MeSH
- DNA-dependentní DNA-polymerasy chemie metabolismus MeSH
- financování organizované MeSH
- HeLa buňky MeSH
- konformace nukleové kyseliny MeSH
- lidé MeSH
- modely genetické MeSH
- multienzymové komplexy chemie metabolismus MeSH
- počítačové zpracování obrazu MeSH
- replikace DNA fyziologie MeSH
- replikon genetika MeSH
- tomografie elektronová MeSH
- Check Tag
- lidé MeSH
We show that DNA double-strand breaks (DSBs) induce complex subcompartmentalization of genome surveillance regulators. Chromatin marked by gamma-H2AX is occupied by ataxia telangiectasia-mutated (ATM) kinase, Mdc1, and 53BP1. In contrast, repair factors (Rad51, Rad52, BRCA2, and FANCD2), ATM and Rad-3-related (ATR) cascade (ATR, ATR interacting protein, and replication protein A), and the DNA clamp (Rad17 and -9) accumulate in subchromatin microcompartments delineated by single-stranded DNA (ssDNA). BRCA1 and the Mre11-Rad50-Nbs1 complex interact with both of these compartments. Importantly, some core DSB regulators do not form cytologically discernible foci. These are further subclassified to proteins that connect DSBs with the rest of the nucleus (Chk1 and -2), that assemble at unprocessed DSBs (DNA-PK/Ku70), and that exist on chromatin as preassembled complexes but become locally modified after DNA damage (Smc1/Smc3). Finally, checkpoint effectors such as p53 and Cdc25A do not accumulate at DSBs at all. We propose that subclassification of DSB regulators according to their residence sites provides a useful framework for understanding their involvement in diverse processes of genome surveillance.
- MeSH
- buněčné linie MeSH
- chromatin fyziologie MeSH
- chromozomální proteiny, nehistonové fyziologie MeSH
- DNA * metabolismus účinky záření MeSH
- fosforylace MeSH
- genom * MeSH
- jaderné proteiny fyziologie MeSH
- kultivované buňky MeSH
- lasery MeSH
- lidé MeSH
- oprava DNA * MeSH
- poškození DNA * MeSH
- protein BRCA1 fyziologie MeSH
- proteinkinasy fyziologie MeSH
- proteiny buněčného cyklu * fyziologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
