Nascent-protein ubiquitination is required for heat shock–induced gene downregulation in human cells

Abstract

hen eukaryotic cells are exposed to environmental stress, they mount an adaptive, conserved and coordinated response at the levels of transcription, translation, cell cycle and metabolism. The transcriptional response called the heat-shock response is typified by upregulation of heat-shock proteins or chaperones driven by the transcription factor heatshock factor (HSF) 1. Initially identified using heat-shock stress, the conserved HSF-driven response is now known to be induced by a variety of cellular conditions 2. Clinical studies have highlighted the relevance of the heat-shock response in cancer and neurodegeneration 3,4. Early studies have shown that stress such as heat shock not only upregulates chaperone genes, but also causes transcriptional downregulation of a handful of genes tested 5,6. Recent genome-wide technologies have confirmed a critical aspect of the heat-shock response: transcriptional downregulation of many highly expressed genes involved in metabolism, protein synthesis and cell cycle 7,8. Stress-induced transcriptional attenuation (SITA) is a rapid process conserved across Drosophila melanogaster 9 , mouse 7 and human cells 10. By downregulating the transcription of key genes, cells probably shift their resources from growth-promoting anabolic activities to dealing with proteotoxic stress. Despite the importance and conserved nature of SITA, relatively little is known about its underlying mechanistic basis. Two key questions need to be addressed: first, how are the rapid and reversible changes in transcription achieved at a molecular level; and second, how is the stress of heat shock sensed and communicated to transcriptional effectors? The primary stress sensor for chaperone upregulation, HSF, is not required for SITA 7 , suggesting an HSF-independent mechanism of stress sensing. How this mechanism is integrated with cellular stress response pathways is not understood. Metazoan transcription proceeds through two highly regulated steps 11,12. First, recruitment of RNA polymerase II (RNA Pol II) to core promoters leads to formation of a pre-initiation complex that transcribes about 20-60 bases downstream of transcription start sites (TSSs) and pauses. Sequence-specific transcription factors along with general transcription factors orchestrate this step. Second, release of promoter-proximal-paused RNA Pol II into productive elongation enables generation of a full-length transcript. Interplay between positive and negative transcription elongation factors (P-TEFs and N-TEFs, respectively) determines the fate of paused RNA Pol II, which is either termination followed by dissociation from promoters or transition to its elongating form. N-TEFs comprise several complexes including negative elongation factor (NELF), DRB-sensitivity inducing factor (DSIF), Integrator, tripartite motif containing 28 (TRIM28) and polymerase-associated factor 1 (PAF1) 13-16. N-TEFs oppose the elongation activity of P-TEFb, which consists of cyclin-dependent kinase 9 (Cdk9) and cyclin T 17. How stress affects the activity ...

NELF Regulates a Promoter-Proximal Step Distinct from RNA Pol II Pause-Release

“…NELF has previously been shown to be dispensable for heat shock-induced gene upregulation in Drosophila cells (Ghosh et al, 2011), which is consistent with our findings. It has also been previously shown that gene downregulation upon heat shock is associated with pausing (Aprile- Garcia et al, 2019;Chen et al, 2017;Mahat et al, 2016a). We found that upon heat shock, RNA Pol II is paused at 1st pause regions for the downregulated genes, consistent with these studies.…”

Aggregation and disaggregation features of the human proteome

“…Aggregation in the nucleus could mean several different things. For example, during stress, proteins have been reported to enter the nucleoli (Frottin et al , 2019) and increase abundance at chromatin (Aprile‐Garcia et al , 2019). These could be linked to our results of increased thermal stability for soluble sub‐pools of aggregators that did not aggregate in heat shock (Fig 5B); the most strongly thermally stabilized proteins were indeed zinc‐finger containing DNA‐binding proteins (Fig 5B).…”

Aggregation and Disaggregation Features of the Human Proteome

“…Aggregation in the nucleus could mean several different things. For example, during stress, proteins have been reported to enter the nucleoli [61] and increase abundance at chromatin [62]. Although the nucleolus is actually proposed to act as a protein quality compartment [61], it is tempting to speculate that chromatin binding could also have protein quality aspects by stabilizing and sequestering unstable DNA-binding proteins during proteotoxic stress.…”