Zooming in: PAGE-Northern Blot Helps to Analyze Anti-Sense Transcripts Originating from Human rIGS under Transcriptional Stress

Ribosomal intergenic spacer (rIGS), located between the 45S rRNA coding arrays in humans, is a deep, unexplored source of small and long non-coding RNA molecules transcribed in certain conditions to help a cell generate a stress response, pass through a differentiation state or fine tune the functioning of the nucleolus as a ribosome biogenesis center of the cell. Many of the non-coding transcripts originating from the rIGS are not characterized to date. Here, we confirm the transcriptional activity of the region laying a 2 kb upstream of the rRNA promoter, and demonstrate its altered expression under transcriptional stress, induced by a wide range of known transcription inhibitors. We managed to show an increased variability of anti-sense transcripts in alpha-amanitin treated cells by applying the low-molecular RNA fraction extracted from agarose gel to PAGE-northern. Also, the fractioning of RNA by size using agarose gel slices occurred, being applicable for determining the sizes of target transcripts via RT-PCR.

Transcriptional Stress Induces Chromatin Relocation of the Nucleotide Excision Repair Factor XPG

Endonuclease XPG participates in nucleotide excision repair (NER), in basal transcription, and in the processing of RNA/DNA hybrids (R-loops): the malfunction of these processes may cause genome instability. Here, we investigate the chromatin association of XPG during basal transcription and after transcriptional stress. The inhibition of RNA polymerase II with 5,6-dichloro-l-β-D-ribofuranosyl benzimidazole (DRB), or actinomycin D (AD), and of topoisomerase I with camptothecin (CPT) resulted in an increase in chromatin-bound XPG, with concomitant relocation by forming nuclear clusters. The cotranscriptional activators p300 and CREB-binding protein (CREBBP), endowed with lysine acetyl transferase (KAT) activity, interact with and acetylate XPG. Depletion of both KATs by RNA interference, or chemical inhibition with C646, significantly reduced XPG acetylation. However, the loss of KAT activity also resulted in increased chromatin association and the relocation of XPG, indicating that these processes were induced by transcriptional stress and not by reduced acetylation. Transcription inhibitors, including C646, triggered the R-loop formation and phosphorylation of histone H2AX (γ-H2AX). Proximity ligation assay (PLA) showed that XPG colocalized with R-loops, indicating the recruitment of the protein to these structures. These results suggest that transcriptional stress-induced XPG relocation may represent recruitment to sites of R-loop processing.

CSN5A Subunit of COP9 Signalosome Is Required for Resetting Transcriptional Stress Memory after Recurrent Heat Stress in Arabidopsis

In nature, plants are exposed to several environmental stresses that can be continuous or recurring. Continuous stress can be lethal, but stress after priming can increase the tolerance of a plant to better prepare for future stresses. Reports have suggested that transcription factors are involved in stress memory after recurrent stress; however, less is known about the factors that regulate the resetting of stress memory. Here, we uncovered a role for Constitutive Photomorphogenesis 5A (CSN5A) in the regulation of stress memory for resetting transcriptional memory genes (APX2 and HSP22) and H3K4me3 following recurrent heat stress. Furthermore, CSN5A is also required for the deposition of H3K4me3 following recurrent heat stress. Thus, CSN5A plays an important role in the regulation of histone methylation and transcriptional stress memory after recurrent heat stress.

The nucleoid-associated protein IHF acts as a ‘transcriptional domainin’ protein coordinating the bacterial virulence traits with global transcription

Bacterial pathogenic growth requires a swift coordination of pathogenicity function with various kinds of environmental stress encountered in the course of host infection. Among the factors critical for bacterial adaptation are changes of DNA topology and binding effects of nucleoid-associated proteins transducing the environmental signals to the chromosome and coordinating the global transcriptional response to stress. In this study, we use the model phytopathogen Dickeya dadantii to analyse the organisation of transcription by the nucleoid-associated heterodimeric protein IHF. We inactivated the IHFα subunit of IHF thus precluding the IHFαβ heterodimer formation and determined both phenotypic effects of ihfA mutation on D. dadantii virulence and the transcriptional response under various conditions of growth. We show that ihfA mutation reorganises the genomic expression by modulating the distribution of chromosomal DNA supercoils at different length scales, thus affecting many virulence genes involved in both symptomatic and asymptomatic phases of infection, including those required for pectin catabolism. Altogether, we propose that IHF heterodimer is a ‘transcriptional domainin’ protein, the lack of which impairs the spatiotemporal organisation of transcriptional stress-response domains harbouring various virulence traits, thus abrogating the pathogenicity of D. dadantii.

p53 activation during ribosome biogenesis regulates normal erythroid differentiation.

The role of ribosome biogenesis in erythroid development is supported by the recognition of erythroid defects in ribosomopathies in both Diamond-Blackfan anemia and 5q- syndrome. Whether ribosome biogenesis exerts a regulatory function on normal erythroid development is still unknown. In the present study, a detailed characterization of ribosome biogenesis dynamics during human and murine erythropoiesis shows that ribosome biogenesis is abruptly interrupted by the drop of rDNA transcription and the collapse of ribosomal protein neo-synthesis. Its premature arrest by RNA polI inhibitor, CX-5461 targets the proliferation of immature erythroblasts. We also show that p53 is activated spontaneously or in response to CX-5461 concomitantly to ribosome biogenesis arrest, and drives a transcriptional program in which genes involved in cell cycle arrest, negative regulation of apoptosis and DNA damage response were upregulated. RNA polI transcriptional stress results in nucleolar disruption and activation of ATR-CHK1-p53 pathway. Our results imply that the timing of ribosome biogenesis extinction and p53 activation are crucial for erythroid development. In ribosomopathies in which ribosome availability is altered by unbalanced production of ribosomal proteins, the threshold of ribosome biogenesis down-regulation could be prematurely reached and together with pathological p53 activation prevents a normal expansion of erythroid progenitors.

Misactivation of multiple starvation responses in yeast by loss of tRNA modifications

Previously, combined loss of different anticodon loop modifications was shown to impair the function of distinct tRNAs in Saccharomyces cerevisiae. Surprisingly, each scenario resulted in shared cellular phenotypes, the basis of which is unclear. Since loss of tRNA modification may evoke transcriptional responses, we characterized global transcription patterns of modification mutants with defects in either tRNAGlnUUG or tRNALysUUU function. We observe that the mutants share inappropriate induction of multiple starvation responses in exponential growth phase, including derepression of glucose and nitrogen catabolite-repressed genes. In addition, autophagy is prematurely and inadequately activated in the mutants. We further demonstrate that improper induction of individual starvation genes as well as the propensity of the tRNA modification mutants to form protein aggregates are diminished upon overexpression of tRNAGlnUUG or tRNALysUUU, the tRNA species that lack the modifications of interest. Hence, our data suggest that global alterations in mRNA translation and proteostasis account for the transcriptional stress signatures that are commonly triggered by loss of anticodon modifications in different tRNAs.

A cellular stress response induced by the CRISPR/dCas9 activation system is not heritable through cell divisions

ABSTRACTThe CRISPR/Cas9 system can be modified to perform ‘epigenetic editing’ by utilizing the catalytically-inactive (dead) Cas9 (dCas9) to recruit regulatory proteins to specific genomic locations. In prior studies, epigenetic editing with multimers of the transactivator VP16 and guide RNAs (gRNAs) was found to cause adverse cellular responses. These side effects may confound studies inducing new cellular properties, especially if the cellular responses are maintained through cell divisions - an epigenetic regulatory property. Here we show how distinct components of this CRISPR/dCas9 activation system, particularly untargeted gRNAs, upregulate genes associated with transcriptional stress, defense response, and regulation of cell death. Our results highlight a previously undetected acute stress response to CRISPR/dCas9 components in human cells, which is transient and not maintained through cell divisions.