chromatin organization
Dotaz
Zobrazit nápovědu
Cells belonging to the germ lineage segregate physically and molecularly from their somatic neighbors during embryogenesis. While germ line-specific chromatin modifications have been identified at later stages in the Caenorhabditis elegans nematode, none have been found in the single P4 germ line founder cell that arises at the beginning of gastrulation. Using light and electron microscopy, we now report that the chromatin organization in the germ line founder cell of the early C. elegans embryo is distinct from that in the neighboring somatic cells. This unique organization is characterized by a greater chromatin compaction and an expansion of the interchromatin compartment. The ultrastructure of individual chromatin domains does not differ between germ line and somatic cells, pointing to a specific organization mainly at the level of the whole nucleus. We show that this higher order reorganization of chromatin is not a consequence of the P4 nucleus being smaller than somatic nuclei or having initiated mitosis. Imaging of living embryos expressing fluorescent markers for both chromatin and P granules revealed that the appearance of a distinct chromatin organization in the P4 cell occurs approximately 10 min after its birth and coincides with the aggregation of P granules around the nucleus, suggesting a possible link between these two events. The higher order reorganization of chromatin that is reported here occurs during the establishment of definitive germ cell identity. The changes we have observed could therefore be a prerequisite for the programming of chromatin totipotency.
- MeSH
- Caenorhabditis elegans embryologie ultrastruktura MeSH
- chromatin metabolismus ultrastruktura MeSH
- embryo nesavčí metabolismus ultrastruktura MeSH
- restrukturace chromatinu fyziologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
This Special Issue highlights the advantages of using combined approaches to explore chromatin molecular complexes [...].
- MeSH
- chromatin * genetika MeSH
- genom * MeSH
- restrukturace chromatinu MeSH
- Publikační typ
- úvodníky MeSH
Chromatin compaction mediates progenitor to post-mitotic cell transitions and modulates gene expression programs, yet the mechanisms are poorly defined. Snf2h and Snf2l are ATP-dependent chromatin remodelling proteins that assemble, reposition and space nucleosomes, and are robustly expressed in the brain. Here we show that mice conditionally inactivated for Snf2h in neural progenitors have reduced levels of histone H1 and H2A variants that compromise chromatin fluidity and transcriptional programs within the developing cerebellum. Disorganized chromatin limits Purkinje and granule neuron progenitor expansion, resulting in abnormal post-natal foliation, while deregulated transcriptional programs contribute to altered neural maturation, motor dysfunction and death. However, mice survive to young adulthood, in part from Snf2l compensation that restores Engrailed-1 expression. Similarly, Purkinje-specific Snf2h ablation affects chromatin ultrastructure and dendritic arborization, but alters cognitive skills rather than motor control. Our studies reveal that Snf2h controls chromatin organization and histone H1 dynamics for the establishment of gene expression programs underlying cerebellar morphogenesis and neural maturation.
- MeSH
- adenosintrifosfatasy metabolismus MeSH
- analýza rozptylu MeSH
- bromodeoxyuridin MeSH
- chromatinová imunoprecipitace MeSH
- chromozomální proteiny, nehistonové metabolismus MeSH
- fluorescence MeSH
- galaktosidy MeSH
- histony metabolismus MeSH
- homeodoménové proteiny metabolismus MeSH
- hybridizace in situ MeSH
- imunohistochemie MeSH
- indoly MeSH
- koncové značení zlomů DNA in situ MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- metoda rotující tyčky MeSH
- mikročipová analýza MeSH
- morfogeneze genetika fyziologie MeSH
- mozeček embryologie MeSH
- myši transgenní MeSH
- myši MeSH
- nervové kmenové buňky metabolismus fyziologie MeSH
- počítačové zpracování obrazu MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- Purkyňovy buňky metabolismus MeSH
- restrukturace chromatinu fyziologie MeSH
- toloniumchlorid MeSH
- transmisní elektronová mikroskopie MeSH
- vývojová regulace genové exprese genetika fyziologie MeSH
- western blotting MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Analysis of histone variants and epigenetic marks is dominated by genome-wide approaches in the form of chromatin immunoprecipitation-sequencing (ChIP-seq) and related methods. Although uncontested in their value for single-copy genes, mapping the chromatin of DNA repeats is problematic for biochemical techniques that involve averaging of cell populations or analysis of clusters of tandem repeats in a single-cell analysis. Extending chromatin and DNA fibers allows us to study the epigenetics of individual repeats in their specific chromosomal context, and thus constitutes an important tool for gaining a complete understanding of the epigenetic organization of genomes. We report that using an optimized fiber extension protocol is essential in order to obtain more reproducible data and to minimize the clustering of fibers. We also demonstrate that the use of super-resolution microscopy is important for reliable evaluation of the distribution of histone modifications on individual fibers. Furthermore, we introduce a custom script for the analysis of methylation levels on DNA fibers and apply it to map the methylation of telomeres, ribosomal genes and centromeres.
Methods in enzymology ; Vol. 304
[1st ed.] xxxiv, 815 s. : ill.
The three-dimensional (3D) organization of chromatin plays a crucial role in the regulation of gene expression. Chromatin conformation is strongly affected by the composition, structural features and dynamic properties of the nucleosome, which in turn determine the nature and geometry of interactions that can occur between neighboring nucleosomes. Understanding how chromatin is spatially organized above the nucleosome level is thus essential for understanding how gene regulation is achieved. Towards this end, great effort has been made to understand how an array of nucleosomes folds into a regular chromatin fiber. This review summarizes new insights into the 3D structure of the chromatin fiber that were made possible by recent advances in cryo-electron microscopy.
Recent studies have shown that histone code dictates the type and structure of chromatin. Bearing in mind the importance of A-type lamins for chromatin arrangement, we studied the effect of trichostatin A (TSA)-induced histone hyperacetylation in lamin A/C-deficient (LMNA-/-) fibroblasts. Lamin A/C deficiency caused condensation of chromosome territories and the nuclear reorganization of centromeric heterochromatin, which was accompanied by the appearance of a chain-like morphology of HP1beta foci. Conversely, histone deacetylase (HDAC) inhibition induced de-condensation of chromosome territories, which compensated the effect of lamin A/C deficiency on chromosome regions. The amount of heterochromatin in the area associated with the nuclear membrane was significantly reduced in LMNA-/- cells when compared with lamin A/C-positive (LMNA+/+) fibroblasts. TSA also decreased the amount of peripheral heterochromatin, similarly as lamin A/C deficiency. In both LMNA+/+ and LMNA-/- cells, physically larger chromosomes were positioned more peripherally as compared with the smaller ones, even after TSA treatment. Our observations indicate that lamin A/C deficiency causes not only reorganization of chromatin and some chromatin-associated domains, but also has an impact on the extent of chromosome condensation. As HDAC inhibition can compensate the lamin A/C-dependent chromatin changes, the interaction between lamins and specifically modified histones may play an important role in higher-order chromatin organization, which influences transcriptional activity.
- MeSH
- acetylace účinky léků MeSH
- buněčné jádro metabolismus MeSH
- centromera metabolismus MeSH
- chromatin metabolismus účinky léků MeSH
- fibroblasty metabolismus MeSH
- financování organizované MeSH
- heterochromatin metabolismus MeSH
- inhibitory enzymů farmakologie MeSH
- inhibitory histondeacetylas MeSH
- jaderné proteiny metabolismus MeSH
- kyseliny hydroxamové farmakologie MeSH
- lamin typ A genetika metabolismus nedostatek MeSH
- lamin typ B metabolismus MeSH
- myši MeSH
- restrukturace chromatinu účinky léků MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
The establishment of long-lasting immunity against pathogens is facilitated by the germinal center (GC) reaction, during which B cells increase their antibody affinity and differentiate into antibody-secreting cells (ASC) and memory cells. These events involve modifications in chromatin packaging that orchestrate the profound restructuring of gene expression networks that determine cell fate. While several chromatin remodelers were implicated in lymphocyte functions, less is known about SMARCA5. Here, using ribosomal pull-down for analyzing translated genes in GC B cells, coupled with functional experiments in mice, we identified SMARCA5 as a key chromatin remodeler in B cells. While the naive B cell compartment remained unaffected following conditional depletion of Smarca5, effective proliferation during B cell activation, immunoglobulin class switching, and as a result GC formation and ASC differentiation were impaired. Single-cell multiomic sequencing analyses revealed that SMARCA5 is crucial for facilitating the transcriptional modifications and genomic accessibility of genes that support B cell activation and differentiation. These findings offer novel insights into the functions of SMARCA5, which can be targeted in various human pathologies.
- MeSH
- adenosintrifosfatasy MeSH
- aktivace lymfocytů imunologie MeSH
- B-lymfocyty * metabolismus imunologie MeSH
- buněčná diferenciace * MeSH
- chromozomální proteiny, nehistonové * metabolismus genetika MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- přesmyk imunoglobulinových tříd genetika MeSH
- restrukturace chromatinu * MeSH
- zárodečné centrum lymfatické uzliny * imunologie metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The nuclear periphery (NP) plays a substantial role in chromatin organization. Heterochromatin at the NP is interspersed with active chromatin surrounding nuclear pore complexes (NPCs); however, details of the peripheral chromatin organization are missing. To discern the distribution of epigenetic marks at the NP of HeLa nuclei, we used structured illumination microscopy combined with a new MATLAB software tool for automatic NP and NPC detection, measurements of fluorescent intensity and statistical analysis of measured data. Our results show that marks for both active and non-active chromatin associate differentially with NPCs. The incidence of heterochromatin marks, such as H3K27me2 and H3K9me2, was significantly lower around NPCs. In contrast, the presence of marks of active chromatin such as H3K4me2 was only decreased very slightly around the NPCs or not at all (H3K9Ac). Interestingly, the histone demethylases LSD1 (also known as KDM1A) and KDM2A were enriched within the NPCs, suggesting that there was a chromatin-modifying mechanism at the NPCs. Inhibition of transcription resulted in a larger drop in the distribution of H1, H3K9me2 and H3K23me2, which implies that transcription has a role in the organization of heterochromatin at the NP.
- MeSH
- buněčné jádro metabolismus MeSH
- chromatin chemie metabolismus MeSH
- epigeneze genetická MeSH
- fluorescenční mikroskopie MeSH
- HeLa buňky MeSH
- heterochromatin chemie MeSH
- histondemethylasy metabolismus MeSH
- histony chemie MeSH
- jaderný obal metabolismus MeSH
- jaderný pór metabolismus MeSH
- lidé MeSH
- mikroskopie metody MeSH
- software MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
