8 research outputs found
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A stable mode of bookmarking by TBP recruits RNA polymerase II to mitotic chromosomes.
Maintenance of transcription programs is challenged during mitosis when chromatin becomes condensed and transcription is silenced. How do the daughter cells re-establish the original transcription program? Here, we report that the TATA-binding protein (TBP), a key component of the core transcriptional machinery, remains bound globally to active promoters in mouse embryonic stem cells during mitosis. Using live-cell single-molecule imaging, we observed that TBP mitotic binding is highly stable, with an average residence time of minutes, in stark contrast to typical TFs with residence times of seconds. To test the functional effect of mitotic TBP binding, we used a drug-inducible degron system and found that TBP promotes the association of RNA Polymerase II with mitotic chromosomes, and facilitates transcriptional reactivation following mitosis. These results suggest that the core transcriptional machinery promotes efficient transcription maintenance globally
Recommended from our members
A stable mode of bookmarking by TBP recruits RNA polymerase II to mitotic chromosomes.
Maintenance of transcription programs is challenged during mitosis when chromatin becomes condensed and transcription is silenced. How do the daughter cells re-establish the original transcription program? Here, we report that the TATA-binding protein (TBP), a key component of the core transcriptional machinery, remains bound globally to active promoters in mouse embryonic stem cells during mitosis. Using live-cell single-molecule imaging, we observed that TBP mitotic binding is highly stable, with an average residence time of minutes, in stark contrast to typical TFs with residence times of seconds. To test the functional effect of mitotic TBP binding, we used a drug-inducible degron system and found that TBP promotes the association of RNA Polymerase II with mitotic chromosomes, and facilitates transcriptional reactivation following mitosis. These results suggest that the core transcriptional machinery promotes efficient transcription maintenance globally
Orlistat and antisense-miRNA-loaded PLGA-PEG nanoparticles for enhanced triple negative breast cancer therapy
Engineering Intracellularly Retained Gaussia Luciferase Reporters for Improved Biosensing and Molecular Imaging Applications
Gaussia
luciferase (GLUC) is a bioluminescent reporter protein
of increasing importance. As a secretory protein, it has increased
sensitivity <i>in vitro</i> and <i>in vivo</i> (∼20 000-fold, and ∼1000-fold, respectively)
over its competitor, secreted alkaline phosphatase. Unfortunately,
this same advantageous secretory nature of GLUC limits its usefulness
for many other possible intracellular applications, <i>e.g.</i>, imaging signaling pathways in intact cells, <i>in vivo</i> imaging, and in developing molecular imaging biosensors to study
protein–protein interactions and protein folding. Hence, to
widen the research applications of GLUC, we developed engineered variants
that increase its intracellular retention both by modifying the N-terminal
secretory signal peptide and by tagging additional sequences to its
C-terminal region. We found that when GLUC was expressed in mammalian
cells, its N-terminal secretory signal peptide comprising amino acids
1–16 was essential for GLUC folding and functional activity
in addition to its inherent secretory property. Modification of the
C-terminus of GLUC by tagging a four amino acid (KDEL) endoplasmic
reticulum targeting peptide in multiple repeats significantly improved
its intracellular retention, with little impact on its folding and
enzymatic activity. We used stable cells expressing this engineered
GLUC with KDEL repeats to monitor chemically induced endoplasmic reticulum
stress on cells. Additionally, we engineered an apoptotic sensor using
modified variants of GLUC containing a four amino acid caspase substrate
peptide (DEVD) between the GLUC protein and the KDEL repeats. Its
use in cell culture resulted in increased GLUC secretion in the growth
medium when cells were treated with the chemotherapeutic drugs doxorubicin,
paclitaxel, and carboplatin. We thus successfully engineered a new
variant GLUC protein that is retained inside cells rather than secreted
extracellularly. We validated this novel reporter by incorporating
it in biosensors for detection of cellular endoplasmic reticulum stress
and caspase activation. This new molecularly engineered enzymatic
reporter has the potential for widespread applications in biological
research