No abstract

No abstract

No 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.

No abstract