scholarly journals JASPer controls interphase histone H3S10 phosphorylation by chromosomal kinase JIL-1 in Drosophila

2019 ◽  
Author(s):  
Christian Albig ◽  
Chao Wang ◽  
Geoffrey P. Dann ◽  
Felix Wojcik ◽  
Tamás Schauer ◽  
...  
Keyword(s):  
Active Chromatin ◽  
Pwwp Domain ◽  
Heterochromatin Formation ◽  
H3s10 Phosphorylation ◽  
Major Form ◽  
Transcriptional Output ◽  
Active Genes

AbstractIn flies, the chromosomal kinase JIL-1 is responsible for most interphase histone H3S10 phosphorylation and has been proposed to protect active chromatin from acquiring heterochromatic marks, like dimethylated histone H3K9 (H3K9me2) and HP1. Here, we show that JIL-1’s targeting to chromatin depends on a new PWWP domain-containing protein JASPer (JIL-1 Anchoring and Stabilizing Protein). The JASPer-JIL-1 (JJ)-complex is the major form of the kinase in vivo and is targeted to active genes and telomeric transposons via binding of the PWWP domain of JASPer to H3K36me3 nucleosomes. Put in place, the complex modulates the transcriptional output. JIL-1 and JJ-complex depletion in cycling cells lead to small changes in H3K9me2 distribution at active genes and telomeric transposons. Finally, we identified many new interactors of the endogenous JJ-complex and propose that JIL-1 not only prevents heterochromatin formation, but also coordinates chromatin-based regulation in the transcribed part of the genome.

scholarly journals JASPer controls interphase histone H3S10 phosphorylation by chromosomal kinase JIL-1 in Drosophila

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Christian Albig ◽  
Chao Wang ◽  
Geoffrey P. Dann ◽  
Felix Wojcik ◽  
Tamás Schauer ◽  
...  
Keyword(s):  
Active Chromatin ◽  
Pwwp Domain ◽  
Heterochromatin Formation ◽  
H3s10 Phosphorylation ◽  
Major Form ◽  
Transcriptional Output ◽  
Active Genes

AbstractIn flies, the chromosomal kinase JIL-1 is responsible for most interphase histone H3S10 phosphorylation and has been proposed to protect active chromatin from acquiring heterochromatic marks, such as dimethylated histone H3K9 (H3K9me2) and HP1. Here, we show that JIL-1’s targeting to chromatin depends on a PWWP domain-containing protein JASPer (JIL-1 Anchoring and Stabilizing Protein). JASPer-JIL-1 (JJ)-complex is the major form of kinase in vivo and is targeted to active genes and telomeric transposons via binding of the PWWP domain of JASPer to H3K36me3 nucleosomes, to modulate transcriptional output. JIL-1 and JJ-complex depletion in cycling cells lead to small changes in H3K9me2 distribution at active genes and telomeric transposons. Finally, we identify interactors of the endogenous JJ-complex and propose that JIL-1 not only prevents heterochromatin formation but also coordinates chromatin-based regulation in the transcribed part of the genome.

scholarly journals Injection of antibodies into the nucleus of amphibian oocytes: an experimental means of interfering with gene expression in the living cell

Development ◽  
1986 ◽  
Vol 97 (Supplement) ◽  
pp. 223-242
Author(s):  
Ulrich Scheer
Keyword(s):  
Living Cell ◽  
Biochemical Analysis ◽  
A Priori ◽  
In Vitro Assays ◽  
Active Chromatin ◽  
Nuclear Antigens ◽  
Experimental Approaches ◽  
Active Genes

The chromatin constituents associated with transcriptionally active genes can be studied by a variety of experimental approaches. Most of the methods used involve the isolation of active chromatin fractions followed by biochemical analysis, or are based on in vitro assays with the ultimate goal of reconstituting faithful transcription by adding purified and defined components to the transcriptional machinery (for reviews see Mathis, Oudet & Chambon, 1980; Rose, Stetler & Jacob, 1983a; Beroldingen et al. 1984; Reeves, 1984; Dynan & Tjian, 1985; Sentenac, 1985). Since these approaches require isolation of chromatin or of individual components therefrom, possible structural rearrangements or selective losses of certain constituents cannot a priori be excluded. Therefore it often remains questionable whether the results obtained in vitro reflect the actual situation in the living cell. In order to examine the nature of transcriptionally active chromatin in the living cell and to decipher the in vivo function of nuclear proteins in transcriptional processes, we have microinjected antibodies to various nuclear antigens directly into the nucleus of living amphibian oocytes.

scholarly journals dP75 safeguards oogenesis by preventing H3K9me2 spreading

2019 ◽  
Author(s):  
Kun Dou ◽  
Yanchao Liu ◽  
Yingpei Zhang ◽  
Chenhui Wang ◽  
Ying Huang ◽  
...  
Keyword(s):  
Physiological Function ◽  
Extensive Study ◽  
Host Factor ◽  
Molecular Function ◽  
Active Chromatin ◽  
Pwwp Domain ◽  
C Terminus ◽  
Histone Kinase ◽  
Hiv Integration

ABSTRACTServing as a host factor for HIV integration, LEDGF/p75 has been under extensive study as a potential target for therapy. However, as a highly conserved protein, its physiological function remains to be thoroughly elucidated. Here we characterize the molecular function of dP75, the Drosophila homolog of p75, during oogenesis. dP75 binds to transcriptionally active chromatin with its PWWP domain. The C-terminus IBD domain-containing region of dP75 physically interacts with the histone kinase Jil-1 and stabilizes it in vivo. Together with Jil-1, dP75 prevents the spreading of the heterochromatin mark–H3K9me2–onto genes required for oogenesis and piRNA production. Without dP75, ectopically silencing of these genes disrupts oogenesis, activates transposons, and causes animal sterility. We propose that dP75, the homolog of an HIV host factor in Drosophila, partners with Jil-1 to ensure gene expression during oogenesis by preventing ectopic heterochromatin spreading.

scholarly journals Force generation by protein–DNA co-condensation

2021 ◽  
Author(s):  
Thomas Quail ◽  
Stefan Golfier ◽  
Maria Elsner ◽  
Keisuke Ishihara ◽  
Vasanthanarayan Murugesan ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Self Assembly ◽  
Spider Silk ◽  
Regulatory Elements ◽  
Heterochromatin Formation ◽  
First Order Phase Transition ◽  
Dna Strand ◽  
First Order Phase

AbstractInteractions between liquids and surfaces generate forces1,2 that are crucial for many processes in biology, physics and engineering, including the motion of insects on the surface of water3, modulation of the material properties of spider silk4 and self-assembly of microstructures5. Recent studies have shown that cells assemble biomolecular condensates via phase separation6. In the nucleus, these condensates are thought to drive transcription7, heterochromatin formation8, nucleolus assembly9 and DNA repair10. Here we show that the interaction between liquid-like condensates and DNA generates forces that might play a role in bringing distant regulatory elements of DNA together, a key step in transcriptional regulation. We combine quantitative microscopy, in vitro reconstitution, optical tweezers and theory to show that the transcription factor FoxA1 mediates the condensation of a protein–DNA phase via a mesoscopic first-order phase transition. After nucleation, co-condensation forces drive growth of this phase by pulling non-condensed DNA. Altering the tension on the DNA strand enlarges or dissolves the condensates, revealing their mechanosensitive nature. These findings show that DNA condensation mediated by transcription factors could bring distant regions of DNA into close proximity, suggesting that this physical mechanism is a possible general regulatory principle for chromatin organization that may be relevant in vivo.

scholarly journals Nuclear condensates of the Polycomb protein chromobox 2 (CBX2) assemble through phase separation

2018 ◽  
Vol 294 (5) ◽  
pp. 1451-1463 ◽  
Author(s):  
Roubina Tatavosian ◽  
Samantha Kent ◽  
Kyle Brown ◽  
Tingting Yao ◽  
Huy Nguyen Duc ◽  
...  
Keyword(s):  
Phase Separation ◽  
Polycomb Group ◽  
Site Directed Mutagenesis ◽  
Developmental Defects ◽  
Heterochromatin Formation ◽  
Intrinsically Disordered ◽  
Polycomb Protein ◽  
Condensate Formation

Polycomb group (PcG) proteins repress master regulators of development and differentiation through organization of chromatin structure. Mutation and dysregulation of PcG genes cause developmental defects and cancer. PcG proteins form condensates in the cell nucleus, and these condensates are the physical sites of PcG-targeted gene silencing via formation of facultative heterochromatin. However, the physiochemical principles underlying the formation of PcG condensates remain unknown, and their determination could shed light on how these condensates compact chromatin. Using fluorescence live-cell imaging, we observed that the Polycomb repressive complex 1 (PRC1) protein chromobox 2 (CBX2), a member of the CBX protein family, undergoes phase separation to form condensates and that the CBX2 condensates exhibit liquid-like properties. Using site-directed mutagenesis, we demonstrated that the conserved residues of CBX2 within the intrinsically disordered region (IDR), which is the region for compaction of chromatin in vitro, promote the condensate formation both in vitro and in vivo. We showed that the CBX2 condensates concentrate DNA and nucleosomes. Using genetic engineering, we report that trimethylation of Lys-27 at histone H3 (H3K27me3), a marker of heterochromatin formation produced by PRC2, had minimal effects on the CBX2 condensate formation. We further demonstrated that the CBX2 condensate formation does not require CBX2–PRC1 subunits; however, the condensate formation of CBX2–PRC1 subunits depends on CBX2, suggesting a mechanism underlying the assembly of CBX2–PRC1 condensates. In summary, our results reveal that PcG condensates assemble through liquid–liquid phase separation (LLPS) and suggest that phase-separated condensates can organize PcG-bound chromatin.

scholarly journals Somatic transposition in the Drosophila intestine occurs in active chromatin and is associated with tumor suppressor gene inactivation

2020 ◽  
Author(s):  
Katarzyna Siudeja ◽  
Marius van den Beek ◽  
Nick Riddiford ◽  
Benjamin Boumard ◽  
Annabelle Wurmser ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Small Rna ◽  
Suppressor Gene ◽  
Somatic Tissue ◽  
Small Rna Sequencing ◽  
Sequencing Data ◽  
Active Chromatin ◽  
Tumor Suppressor Gene Inactivation ◽  
Somatic Transposition

AbstractTransposable elements (TEs) play a significant role in evolution by contributing to genetic variation through germline insertional activity. However, how TEs act in somatic cells and tissues is not well understood. Here, we address the prevalence of transposition in a somatic tissue, exploiting the Drosophila midgut as a model system. Using whole-genome sequencing of in vivo clonally expanded gut tissue, we map hundreds of high-confidence somatic TE integration sites genome-wide. We show that somatic retrotransposon insertions are associated with inactivation of the tumor suppressor Notch, likely contributing to neoplasia formation. Moreover, by applying Oxford Nanopore long-read sequencing technology, as well as by mapping germline TE activity, we provide evidence suggesting tissue-specific differences in retrotransposition. By comparing somatic TE insertional activity with transcriptomic and small RNA sequencing data, we demonstrate that transposon mobility cannot be simply predicted by whole tissue TE expression levels or by small RNA pathway activity. Finally, we reveal that somatic TE insertions in the adult fly intestine are found preferentially in genic regions and open, transcriptionally active chromatin. Together, our findings provide clear evidence of ongoing somatic transposition in Drosophila and delineate previously unknown underlying features of somatic TE mobility in vivo.

Formaldehyde cross-linking and immunoprecipitation demonstrate developmental changes in H1 association with transcriptionally active genes

1991 ◽  
Vol 11 (3) ◽  
pp. 1729-1733
Author(s):  
P C Dedon ◽  
J A Soults ◽  
C D Allis ◽  
M A Gorovsky
Keyword(s):  
Transcriptional Activity ◽  
Inverse Correlation ◽  
Dna Interaction ◽  
Histone H1 ◽  
Developmental Changes ◽  
Cross Linking ◽  
Developmental States ◽  
Logarithmic Growth ◽  
Active Genes

The in vivo association of histone H1 with specific genes in Tetrahymena thermophila was studied by using a simplified cross-linking and immunoprecipitation technique. Four genes were analyzed whose activities vary in three different developmental states (logarithmic growth, starvation, and conjugation). Hybridization of the immunoprecipitated DNA to cloned probes showed an inverse correlation between the level of immunoprecipitation with H1 antiserum and transcriptional activity. This represents the first demonstration of an alteration in histone H1-DNA interaction associated with developmental changes in transcriptional activity.

scholarly journals The 3′ processing of antisense RNAs physically links to chromatin-based transcriptional control

2020 ◽  
Vol 117 (26) ◽  
pp. 15316-15321 ◽  
Author(s):  
Xiaofeng Fang ◽  
Zhe Wu ◽  
Oleg Raitskin ◽  
Kimberly Webb ◽  
Philipp Voigt ◽  
...  
Keyword(s):  
Noncoding Rna ◽  
Transcriptional Control ◽  
Rna Binding ◽  
Antisense Transcription ◽  
Set Domain ◽  
Antisense Transcripts ◽  
Polycomb Repressive Complex 2 ◽  
Active Transcription ◽  
Transcriptional Output

Noncoding RNA plays essential roles in transcriptional control and chromatin silencing. AtArabidopsis thaliana FLC,antisense transcription quantitatively influences transcriptional output, but the mechanism by which this occurs is still unclear. Proximal polyadenylation of the antisense transcripts by FCA, an RNA-binding protein that physically interacts with RNA 3′ processing factors, reducesFLCtranscription. This process genetically requires FLD, a homolog of the H3K4 demethylase LSD1. However, the mechanism linking RNA processing to FLD function had not been established. Here, we show that FLD tightly associates with LUMINIDEPENDENS (LD) and SET DOMAIN GROUP 26 (SDG26) in vivo, and, together, they prevent accumulation of monomethylated H3K4 (H3K4me1) over theFLCgene body. SDG26 interacts with the RNA 3′ processing factor FY (WDR33), thus linking activities for proximal polyadenylation of the antisense transcripts to FLD/LD/SDG26-associated H3K4 demethylation. We propose this demethylation antagonizes an active transcription module, thus reducing H3K36me3 accumulation and increasing H3K27me3. Consistent with this view, we show that Polycomb Repressive Complex 2 (PRC2) silencing is genetically required by FCA to repressFLC. Overall, our work provides insights into RNA-mediated chromatin silencing.

scholarly journals Gamma rays and bleomycin nick DNA and reverse the DNase I sensitivity of beta-globin gene chromatin in vivo.

1987 ◽  
Vol 7 (5) ◽  
pp. 1917-1924 ◽  
Author(s):  
B Villeponteau ◽  
H G Martinson
Keyword(s):  
Globin Gene ◽  
Dnase I ◽  
Globin Genes ◽  
Torsional Stress ◽  
Beta Globin ◽  
Cell Penetration ◽  
Dnase I Sensitivity ◽  
Chicken Erythrocytes ◽  
Active Genes

The active beta-globin genes in chicken erythrocytes, like all active genes, reside in large chromatin domains which are preferentially sensitive to digestion by DNase I. We have recently proposed that the special structure of chromatin in active domains is maintained by torsional stress in the DNA (Villeponteau et al., Cell 39:469-478, 1984). This hypothesis predicts that nicking of the DNA within any such chromosomal domain in vivo will relax the DNA and lead to loss of the special DNase I-sensitive state. Here we have tested this prediction by using gamma irradiation and bleomycin treatment to cleave DNA within intact chicken embryo erythrocytes. Both treatments cause reversal of DNase I sensitivity. Moreover, reversal occurs at approximately one nick per 150 kilobase pairs for both agents despite their entirely unrelated modes of cell penetration and DNA attack. These results suggest that the domain of DNase I sensitivity surrounding the beta-globin genes comprises 150 kilobase pairs of chromatin under torsional stress and that a single DNA nick in this region is sufficient to reverse the DNase I sensitivity throughout the entire domain.

scholarly journals Theory of active chromatin remodeling

2019 ◽  
Author(s):  
Zhongling Jiang ◽  
Bin Zhang
Keyword(s):  
Chromatin Remodeling ◽  
Computational Models ◽  
Energy Landscape ◽  
Nucleosome Positioning ◽  
Active Chromatin ◽  
Intrinsic Signals ◽  
Biologically Relevant ◽  
The Impact ◽  
Prediction In Silico

Nucleosome positioning controls the accessible regions of chromatin and plays essential roles in DNA-templated processes. ATP driven remodeling enzymes are known to be crucial for its establishment in vivo, but their non-equilibrium nature has hindered the development of a unified theoretical framework for nucleosome positioning. Using a perturbation theory, we show that the effect of these enzymes can be well approximated by effective equilibrium models with rescaled temperatures and interactions. Numerical simulations support the accuracy of the theory in predicting both kinetic and steady-state quantities, including the effective temperature and the radial distribution function, in biologically relevant regimes. The energy landscape view emerging from our study provides an intuitive understanding for the impact of remodeling enzymes in either reinforcing or overwriting intrinsic signals for nucleosome positioning, and may help improve the accuracy of computational models for its prediction in silico.

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