scholarly journals Drosophila DAxud1: A New Element in Transcriptional Pausing Complex Stabilization

2021 ◽  
Author(s):  
JM Zuñiga-Hernández ◽  
C Meneses ◽  
M Bastías ◽  
ML Allende ◽  
Alvaro Glavic
Keyword(s):  
Heat Shock ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Polytene Chromosome ◽  
Chromatin Immunoprecipitation ◽  
Environmental Changes ◽  
Transcriptional Response ◽  
Cellular Systems ◽  
Gene Group ◽  
Transcriptional Pausing

Abstract BackgroundA rapid transcriptional response under an acute stimulus is common in all cellular systems and is an adaptation that allows tolerance to environmental changes. A gene group that has been studied because of its fast response and cytoprotective effects are the hsp genes (encodingHeat Shock Proteins(HSPs), conserved chaperones).. Under normal conditions, the mRNA and protein levels of the main hsp genes are low but they increase rapidly upon heat shock (HS). This is achieved due to the presence of an RNA Polymerase II pausing complex located +30-50 bp from TSS. This complex maintains a partially synthesized RNA strand of said length, poised to resume synthesis, and undergoes subsequent transcriptional inactivation to restore transcript levels after environmental stabilization.MethodsThe Gal4/UAS system was used to modify dAxud1 expression in a tissue specific manner. A DAxud1-GFP fusion was expressed in salivary glands to perform polytene chromosome immunofluorescence and chromatin immunoprecipitation. DAxud1 genome occupancy data was achieved expressing Dam-DAxud1 in imaginal wing discs using Gal4/UAS (TaDa-seq).ResultsUsing TaDa-seq, we demonstrate that DAxud1 protein is present mainly near the TSS of significant occupied genes, most frequently in the first intron. This results also revealed DAxud1 is present in hsp genes, mainly in promoter zone. Following these results, we found that, under dAxud knockdown, larvae and adults flies have a diminished thermotolerance, despite showing an increase in hsp transcripts in larval tissues. We performed polytene chromosome immunofluorescence for DAxud1-GFP, revealing extensive, but dynamic localization on chromatin in hsp70 loci. This was confirmed with chromatin immunoprecipitation. We also found that DAxud1 overexpression leads to an enrichment of RNA Polymerase II at the 5’ end of the hsp70 gene, with a decrease in its transcripts. Importantly, we show interaction of DAxud1 with NELF-B, a component of the transcriptional pausing complex, and knockdown of both genes individually has similar effects on hsp70 transcription.ConclusionDAxud1 protein is a component of chromatin, that relocates under stress conditions such as heat shock, playing a role in maintaining RNA Polymerase II stalled at the 5’ of hsp70, possibly through a pausing mechanism based on its interaction with NELF-B.

scholarly journals RNA polymerase II progression through H3K27me3-enriched gene bodies requires JMJD3 histone demethylase

2013 ◽  
Vol 24 (3) ◽  
pp. 351-360 ◽  
Author(s):  
Conchi Estarás ◽  
Raquel Fueyo ◽  
Naiara Akizu ◽  
Sergi Beltrán ◽  
Marian A. Martínez-Balbás
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Chromatin Immunoprecipitation ◽  
Target Genes ◽  
Transforming Growth Factor ◽  
Transcriptional Response ◽  
Transforming Growth Factor Β ◽  
Genome Wide ◽  
Pathway Activation

JMJD3 H3K27me3 demethylase plays an important role in the transcriptional response to different signaling pathways; however, the mechanism by which it facilitates transcription has been unclear. Here we show that JMJD3 regulates transcription of transforming growth factor β (TGFβ)–responsive genes by promoting RNA polymerase II (RNAPII) progression along the gene bodies. Using chromatin immunoprecipitation followed by sequencing experiments, we show that, upon TGFβ treatment, JMJD3 and elongating RNAPII colocalize extensively along the intragenic regions of TGFβ target genes. According to these data, genome-wide analysis shows that JMJD3-dependent TGFβ target genes are enriched in H3K27me3 before TGFβ signaling pathway activation. Further molecular analyses demonstrate that JMJD3 demethylates H3K27me3 along the gene bodies, paving the way for the RNAPII progression. Overall these findings uncover the mechanism by which JMJD3 facilitates transcriptional activation.

scholarly journals The rye Mutants Identify a Role for Ssn/Srb Proteins of the RNA Polymerase II Holoenzyme During Stationary Phase Entry in Saccharomyces cerevisiae

Genetics ◽  
2001 ◽  
Vol 157 (1) ◽  
pp. 17-26 ◽  
Author(s):  
Ya-Wen Chang ◽  
Susie C Howard ◽  
Yelena V Budovskaya ◽  
Jasper Rine ◽  
Paul K Herman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Growth ◽  
Rna Polymerase ◽  
Stationary Phase ◽  
Yeast Cell ◽  
Rna Polymerase Ii ◽  
Transcriptional Response ◽  
Nutrient Deprivation ◽  
Ras Signaling ◽  
Rna Polymerase Ii Holoenzyme

Abstract Saccharomyces cerevisiae cells enter into a distinct resting state, known as stationary phase, in response to specific types of nutrient deprivation. We have identified a collection of mutants that exhibited a defective transcriptional response to nutrient limitation and failed to enter into a normal stationary phase. These rye mutants were isolated on the basis of defects in the regulation of YGP1 expression. In wild-type cells, YGP1 levels increased during the growth arrest caused by nutrient deprivation or inactivation of the Ras signaling pathway. In contrast, the levels of YGP1 and related genes were significantly elevated in the rye mutants during log phase growth. The rye defects were not specific to this YGP1 response as these mutants also exhibited multiple defects in stationary phase properties, including an inability to survive periods of prolonged starvation. These data indicated that the RYE genes might encode important regulators of yeast cell growth. Interestingly, three of the RYE genes encoded the Ssn/Srb proteins, Srb9p, Srb10p, and Srb11p, which are associated with the RNA polymerase II holoenzyme. Thus, the RNA polymerase II holoenzyme may be a target of the signaling pathways responsible for coordinating yeast cell growth with nutrient availability.

Heat shock-induced alterations in phosphorylation of the largest subunit of RNA polymerase II as revealed by monoclonal antibodies CC-3 and MPM-2

1999 ◽  
Vol 77 (4) ◽  
pp. 367-374 ◽  
Author(s):  
Sébastien B Lavoie ◽  
Alexandra L Albert ◽  
Alain Thibodeau ◽  
Michel Vincent
Keyword(s):  
Heat Shock ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Activity ◽  
Phosphorylation Sites ◽  
Stress Genes ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Heat Shocks ◽  
Carboxy Terminal

The phosphorylation of the carboxy-terminal domain of the largest subunit of RNA polymerase II plays an important role in the regulation of transcriptional activity and is also implicated in pre-mRNA processing. Different stresses, such as a heat shock, induce a marked alteration in the phosphorylation of this domain. The expression of stress genes by RNA polymerase II, to the detriment of other genes, could be attributable to such modifications of the phosphorylation sites. Using two phosphodependent antibodies recognizing distinct hyperphosphorylated forms of RNA polymerase II largest subunit, we studied the phosphorylation state of the subunit in different species after heat shocks of varying intensities. One of these antibodies, CC-3, preferentially recognizes the carboxy-terminal domain of the largest subunit under normal conditions, but its reactivity is diminished during stress. In contrast, the other antibody used, MPM-2, demonstrated a strong reactivity after a heat shock in most species studied. Therefore, CC-3 and MPM-2 antibodies discriminate between phosphoisomers that may be functionally different. Our results further indicate that the pattern of phosphorylation of RNA polymerase II in most species varies in response to environmental stress.Key words: RNA polymerase II, heat shock, phosphorylation, CC-3, MPM-2.

scholarly journals Heat-shock inactivation of the TFIIH-associated kinase and change in the phosphorylation sites on the C-terminal domain of RNA polymerase II

1997 ◽  
Vol 25 (4) ◽  
pp. 694-700 ◽  
Author(s):  
M.-F. Dubois ◽  
M. Vincent ◽  
M. Vigneron ◽  
J. Adamczewski ◽  
J.-M. Egly ◽  
...  
Keyword(s):  
Heat Shock ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Phosphorylation Sites ◽  
Terminal Domain

scholarly journals Fcp1 Dephosphorylation of the RNA Polymerase II C-Terminal Domain Is Required for Efficient Transcription of Heat Shock Genes

2012 ◽  
Vol 32 (17) ◽  
pp. 3428-3437 ◽  
Author(s):  
N. J. Fuda ◽  
M. S. Buckley ◽  
W. Wei ◽  
L. J. Core ◽  
C. T. Waters ◽  
...  
Keyword(s):  
Heat Shock ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Heat Shock Genes ◽  
Terminal Domain

scholarly journals Rapid changes in Drosophila transcription after an instantaneous heat shock.

1993 ◽  
Vol 13 (6) ◽  
pp. 3456-3463 ◽  
Author(s):  
T O'Brien ◽  
J T Lis
Keyword(s):  
Heat Shock ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Uv Light ◽  
Hsp70 Gene ◽  
Rapid Induction ◽  
Rapid Changes ◽  
Rapid Activation ◽  
Kinetics Of

Heat shock rapidly activates expression of some genes and represses others. The kinetics of changes in RNA polymerase distribution on heat shock-modulated genes provides a framework for evaluating the mechanisms of activation and repression of transcription. Here, using two methods, we examined the changes in RNA polymerase II association on a set of Drosophila genes at 30-s intervals following an instantaneous heat shock. In the first method, Drosophila Schneider line 2 cells were quickly frozen to halt transcription, and polymerase distribution was analyzed by a nuclear run-on assay. RNA polymerase transcription at the 5' end of the hsp70 gene could be detected within 30 to 60 s of induction, and by 120 s the first wave of polymerase could already be detected near the 3' end of the gene. A similar rapid induction was found for the small heat shock genes (hsp22, hsp23, hsp26, and hsp27). In contrast to this rapid activation, transcription of the histone H1 gene was found to be rapidly repressed, with transcription reduced by approximately 90% within 300 s of heat shock. Similar results were obtained by an in vivo UV cross-linking assay. In this second method, cell samples removed at 30-s intervals were irradiated with 40-microseconds bursts of UV light from a Xenon flash lamp, and the distribution of polymerase was examined by precipitating UV cross-linked protein-DNA complexes with an antibody to RNA polymerase II. Both approaches also showed the in vivo rate of movement of the first wave of RNA polymerase through the hsp70 gene to be approximately 1.2 kb/min.

Correlation between the activity of a 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole-insensitive puff and the synthesis of major heat-shock polypeptide, hsp70, in Chironomus thummi

1988 ◽  
Vol 66 (11) ◽  
pp. 1177-1185 ◽  
Author(s):  
D. Barettino ◽  
G. Morcillo ◽  
J. L. Díez ◽  
M. T. Carretero ◽  
M. J. Carmona
Keyword(s):  
Heat Shock ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Specific Inhibitor ◽  
In Vitro Transcription ◽  
The Other ◽  
Low Salt ◽  
Transcription Inhibitor ◽  
Chironomus Thummi

The induction of puff III-A3b, a major heat-shock puff in Chironomus thummi salivary cells, was insensitive to the transcription inhibitor 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB), whereas no transcriptional activity could be detected at the other heat-shock puffs in the presence of this drug. In these conditions, a polypeptide with the same Mr and isoform pattern as those of the major heat-shock polypeptide, hsp70, was synthesized. These results suggest that hsp70 is encoded by locus III-A3b. In addition to DRB insensitivity, incorporation of [3H]UTP on puff III-A3b took place in an in vitro transcription assay under low-salt conditions (100 mM NaCl); no labelling could be detected at the other heat-shock puffs under these conditions. Although DRB has been reported as a specific inhibitor of RNA polymerase II-directed transcription, and although the low-salt conditions were not propitious for the activity of this enzyme, RNA polymerase II was detected on puff III-A3b and on the other heat-shock puffs by immunofluorescence with anti-RNA polymerase II antibodies.

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