Dynamic coalescence of yeast Heat Shock Protein genes bypasses the requirement for actin

Abstract

Nuclear actin has been implicated in dynamic chromatin rearrangements in diverse eukaryotes. In mammalian cells, it is required to reposition double-strand DNA breaks to enable homologous recombination repair and to enhance transcription by facilitating Pol II recruitment to gene promoters. In the yeast Saccharomyces cerevisiae, nuclear actin modulates interphase chromosome dynamics and is required to reposition the induced INO1 gene to the nuclear periphery. Here we have investigated the role of actin in driving intergenic interactions between Heat Shock Factor 1 (Hsf1)-regulated Heat Shock Protein (HSP) genes in budding yeast. These genes, dispersed on multiple chromosomes, dramatically reposition following exposure of cells to acute thermal stress, leading to their clustering within dynamic biomolecular condensates. Using an auxin-induced degradation strategy, we found that conditional depletion of nucleators of either linear or branched F-actin (Bni1/Bnr1 and Arp2, respectively) had little or no effect on heat shocked-induced HSP gene coalescence or transcription. In addition, we found that pretreatment of cells with latrunculin A (Lat-A), an inhibitor of both filamentous and monomeric actin, failed to affect intergenic interactions between activated HSP genes as well as their heat shock-induced intragenic looping and folding. Moreover, Lat-A pretreatment had little detectable effect on HSP gene expression at either RNA or protein levels. In notable contrast, we confirmed that repositioning of activated INO1 to the nuclear periphery and its proper expression do require actin. Collectively, our work suggests that transcriptional activation and 3D genome restructuring of thermally induced, Hsf1-regulated genes can occur in the absence of actin.

Heat Shock Factor 1 forms nuclear condensates and restructures the yeast genome before activating target genes

“…LRY040 was constructed by transforming LRY037 with an amplicon containing RPB3-mCherry::hphMX6 , obtained using genomic DNA from strain SCY004 (Chowdhary et al, 2022) as template. LRY100 and LRY102 were constructed using LRY016 (Rubio & Gross, 2023) as recipient of the mini-degron tag amplified from pHyg-AID*-9myc (Morawska & Ulrich, 2013), targeted to the C-terminus of HSF1 and RPB1 , respectively.…”

“…RT-qPCR was conducted as previously described using 25 mL cell culture aliquots (Rubio & Gross, 2023). PCR primers used are listed in Supplemental File 1 – Table 4 .…”

“…TaqI-3C was performed as previously described (Rubio & Gross, 2023). A master cell culture was grown at 30°C in YPDA from OD 600 =0.15 to a final OD 600 =0.8.…”

“…LRY100 and LRY102 were constructed using LRY016 (Rubio & Gross, 2023) as recipient of the mini-degron tag amplified from pHyg-AID*-9myc (Morawska & Ulrich, 2013), targeted to the C-terminus of HSF1 and RPB1, respectively.…”

“…An intriguing feature of HSR genes is the fact that they coalesce into discrete intranuclear foci in response to heat shock. Such interactions have been documented using both molecular (chromosome conformation capture (3C)) and imaging (fluorescence microscopy) approaches (Chowdhary et al, 2017(Chowdhary et al, , 2019(Chowdhary et al, , 2022Rubio & Gross, 2023). Intriguingly, physical interactions specifically involve Hsf1 targets irrespective of their location in the genome.…”