scholarly journals KAP1 is an antiparallel dimer with a functional asymmetry

2019 ◽  
Vol 2 (4) ◽  
pp. e201900349 ◽  
Author(s):  
Giulia Fonti ◽  
Maria J Marcaida ◽  
Louise C Bryan ◽  
Sylvain Träger ◽  
Alexandra S Kalantzi ◽  
...  
Keyword(s):  
Single Molecule ◽  
Binding Sites ◽  
Mammalian Cells ◽  
Scattering Data ◽  
Heterochromatin Protein 1 ◽  
Heterochromatin Protein ◽  
Krab Domain ◽  
Functional Implications ◽  
New Gene ◽  
Expression In Mammalian Cells

KAP1 (KRAB domain–associated protein 1) plays a fundamental role in regulating gene expression in mammalian cells by recruiting different transcription factors and altering the chromatin state. In doing so, KAP1 acts both as a platform for macromolecular interactions and as an E3 small ubiquitin modifier ligase. This work sheds light on the overall organization of the full-length protein combining solution scattering data, integrative modeling, and single-molecule experiments. We show that KAP1 is an elongated antiparallel dimer with an asymmetry at the C-terminal domains. This conformation is consistent with the finding that the Really Interesting New Gene (RING) domain contributes to KAP1 auto-SUMOylation. Importantly, this intrinsic asymmetry has key functional implications for the KAP1 network of interactions, as the heterochromatin protein 1 (HP1) occupies only one of the two putative HP1 binding sites on the KAP1 dimer, resulting in an unexpected stoichiometry, even in the context of chromatin fibers.

scholarly journals KAP1 is an antiparallel dimer with a natively functional asymmetry

2019 ◽  
Author(s):  
Giulia Fonti ◽  
Maria J. Marcaida ◽  
Louise C. Bryan ◽  
Sylvain Traeger ◽  
Alexandra S. Kalantzi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Molecule ◽  
Binding Sites ◽  
Mammalian Cells ◽  
Heterochromatin Protein 1 ◽  
Heterochromatin Protein ◽  
Krab Domain ◽  
Sumo Ligase ◽  
Functional Implications ◽  
Expression In Mammalian Cells

AbstractKAP1 (KRAB-domain associated protein 1) plays a fundamental role in regulating gene expression in mammalian cells by recruiting different transcription factors and altering the chromatin state. In doing so, KAP1 acts both as a platform for macromolecular interactions and as an E3 SUMO ligase. This work sheds light on the overall organization of the full-length protein combining solution scattering diffraction data, integrative modeling and single-molecule experiments. We show that KAP1 is an elongated antiparallel dimer with a native asymmetry at the C-terminal domain. This conformation supports our finding that the RING domain contributes to KAP1 auto-SUMOylation. Importantly, this intrinsic asymmetry has key functional implications for the KAP1 network of interactions, as the heterochromatin protein 1 (HP1) occupies only one of the two putative HP1 binding sites on the KAP1 dimer, resulting in an unexpected stoichiometry, even in the context of chromatin fibers.

scholarly journals The Integrity of piRNA Clusters is Abolished by Insulators in the Drosophila Germline

Genes ◽  
2019 ◽  
Vol 10 (3) ◽  
pp. 209 ◽  
Author(s):  
Elizaveta Radion ◽  
Olesya Sokolova ◽  
Sergei Ryazansky ◽  
Pavel Komarov ◽  
Yuri Abramov ◽  
...  
Keyword(s):  
Binding Sites ◽  
Regulatory Elements ◽  
Evolutionary Constraints ◽  
Heterochromatin Protein 1 ◽  
Heterochromatin Protein ◽  
Genome Wide Analysis ◽  
Genome Wide ◽  
Pirna Cluster ◽  
A Genome ◽  
Cluster Activity

Piwi-interacting RNAs (piRNAs) control transposable element (TE) activity in the germline. piRNAs are produced from single-stranded precursors transcribed from distinct genomic loci, enriched by TE fragments and termed piRNA clusters. The specific chromatin organization and transcriptional regulation of Drosophila germline-specific piRNA clusters ensure transcription and processing of piRNA precursors. TEs harbour various regulatory elements that could affect piRNA cluster integrity. One of such elements is the suppressor-of-hairy-wing (Su(Hw))-mediated insulator, which is harboured in the retrotransposon gypsy. To understand how insulators contribute to piRNA cluster activity, we studied the effects of transgenes containing gypsy insulators on local organization of endogenous piRNA clusters. We show that transgene insertions interfere with piRNA precursor transcription, small RNA production and the formation of piRNA cluster-specific chromatin, a hallmark of which is Rhino, the germline homolog of the heterochromatin protein 1 (HP1). The mutations of Su(Hw) restored the integrity of piRNA clusters in transgenic strains. Surprisingly, Su(Hw) depletion enhanced the production of piRNAs by the domesticated telomeric retrotransposon TART, indicating that Su(Hw)-dependent elements protect TART transcripts from piRNA processing machinery in telomeres. A genome-wide analysis revealed that Su(Hw)-binding sites are depleted in endogenous germline piRNA clusters, suggesting that their functional integrity is under strict evolutionary constraints.

scholarly journals The Assembly and Maintenance of Heterochromatin Initiated by Transgene Repeats Are Independent of the RNA Interference Pathway in Mammalian Cells

2006 ◽  
Vol 26 (11) ◽  
pp. 4028-4040 ◽  
Author(s):  
Fangwei Wang ◽  
Naoki Koyama ◽  
Hiroko Nishida ◽  
Tokuko Haraguchi ◽  
Walter Reith ◽  
...  
Keyword(s):  
Rna Interference ◽  
Mammalian Cells ◽  
Cytosine Methylation ◽  
Chromatin Condensation ◽  
Rnai Pathway ◽  
Small Interfering Rnas ◽  
Heterochromatin Protein 1 ◽  
Independent Manner ◽  
Heterochromatin Protein ◽  
Cpg Dinucleotides

ABSTRACT A role for the RNA interference (RNAi) pathway in the establishment of heterochromatin is now well accepted for various organisms. Less is known about its relevance and precise role in mammalian cells. We previously showed that tandem insertion of a 1,000-copy inducible transgene into the genome of baby hamster kidney (BHK) cells initiated the formation of an extremely condensed chromatin locus. Here, we characterized the inactive transgenic locus as heterochromatin, since it was associated with heterochromatin protein 1 (HP1), histone H3 trimethylated at lysine 9, and cytosine methylation in CpG dinucleotides. Northern blot analysis did not detect any transgene-derived small RNAs. RNAi-mediated Dicer knockdown did not disrupt the heterochromatic transgenic locus or up-regulate transgene expression. Moreover, neither Dicer knockdown nor overexpression of transgene-directed small interfering RNAs altered the bidirectional transition of the transgenic locus between the heterochromatic and euchromatic states. Interestingly, tethering of HP1 to the transgenic locus effectively induced transgene silencing and chromatin condensation in a Dicer-independent manner, suggesting a role for HP1 in maintaining the heterochromatic locus. Our results suggest that the RNAi pathway is not required for the assembly and maintenance of noncentromeric heterochromatin initiated by tandem transgene repeats in mammalian cells.

Single-Molecule Imaging of Active Mitochondrial Nitroreductases using a Photo-Crosslinking Fluorescent Sensor

2019 ◽  
Author(s):  
Zacharias Thiel ◽  
Pablo Rivera-Fuentes
Keyword(s):  
Subcellular Localization ◽  
Fluorescent Probe ◽  
Single Molecule ◽  
Mammalian Cells ◽  
Fluorescent Sensor ◽  
Biological Functions ◽  
Localization Microscopy ◽  
Drug Activation ◽  
Nitroreductase Activity ◽  
Single Molecule Localization Microscopy

Many biomacromolecules are known to cluster in microdomains with specific subcellular localization. In the case of enzymes, this clustering greatly defines their biological functions. Nitroreductases are enzymes capable of reducing nitro groups to amines and play a role in detoxification and pro-drug activation. Although nitroreductase activity has been detected in mammalian cells, the subcellular localization of this activity remains incompletely characterized. Here, we report a fluorescent probe that enables super-resolved imaging of pools of nitroreductase activity within mitochondria. This probe is activated sequentially by nitroreductases and light to give a photo-crosslinked adduct of active enzymes. In combination with a general photoactivatable marker of mitochondria, we performed two-color, threedimensional, single-molecule localization microscopy. These experiments allowed us to image the sub-mitochondrial organization of microdomains of nitroreductase activity.<br>

Single-Molecule Imaging of Active Mitochondrial Nitroreductases using a Photo-Crosslinking Fluorescent Sensor

2019 ◽  
Author(s):  
Zacharias Thiel ◽  
Pablo Rivera-Fuentes
Keyword(s):  
Subcellular Localization ◽  
Fluorescent Probe ◽  
Single Molecule ◽  
Mammalian Cells ◽  
Fluorescent Sensor ◽  
Biological Functions ◽  
Localization Microscopy ◽  
Drug Activation ◽  
Nitroreductase Activity ◽  
Single Molecule Localization Microscopy

Many biomacromolecules are known to cluster in microdomains with specific subcellular localization. In the case of enzymes, this clustering greatly defines their biological functions. Nitroreductases are enzymes capable of reducing nitro groups to amines and play a role in detoxification and pro-drug activation. Although nitroreductase activity has been detected in mammalian cells, the subcellular localization of this activity remains incompletely characterized. Here, we report a fluorescent probe that enables super-resolved imaging of pools of nitroreductase activity within mitochondria. This probe is activated sequentially by nitroreductases and light to give a photo-crosslinked adduct of active enzymes. In combination with a general photoactivatable marker of mitochondria, we performed two-color, threedimensional, single-molecule localization microscopy. These experiments allowed us to image the sub-mitochondrial organization of microdomains of nitroreductase activity.<br>

BacMam System for Rapid Recombinant Protein Expression in Mammalian Cells

2019 ◽  
pp. 205-208 ◽  
Author(s):  
Deepak B. Thimiri Govinda Raj ◽  
Niamat Ali Khan ◽  
Srisaran Venkatachalam ◽  
Sivakumar Arumugam
Keyword(s):  
Recombinant Protein ◽  
Protein Expression ◽  
Mammalian Cells ◽  
Recombinant Protein Expression ◽  
Expression In Mammalian Cells
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