Heat shock factors (HSFs) are essential for all organisms to survive exposures to acute stress. They are best known as inducible transcriptional regulators of genes encoding molecular chaperones and other stress proteins. Four members of the HSF family are also important for normal development and lifespan-enhancing pathways, and the repertoire of HSF targets has thus expanded well beyond the heat shock genes. These unexpected observations have uncovered complex layers of post-translational regulation of HSFs that integrate the metabolic state of the cell with stress biology, and in doing so control fundamental aspects of the health of the proteome and ageing.

https://www.auburn.edu/academic/classes/biol/6190/refs/L06/52400148.pdf

Heat shock factors: integrators of cell stress, development and lifespan

The long-term health of the cell is inextricably linked to protein quality control. Under optimal conditions this is accomplished by protein homeostasis, a highly complex network of molecular interactions that balances protein biosynthesis, folding, translocation, assembly/disassembly, and clearance. This review will examine the consequences of an imbalance in homeostasis on the flux of misfolded proteins that, if unattended, can result in severe molecular damage to the cell. Adaptation and survival requires the ability to sense damaged proteins and to coordinate the activities of protective stress response pathways and chaperone networks. Yet, despite the abundance and apparent capacity of chaperones and other components of homeostasis to restore folding equilibrium, the cell appears poorly adapted for chronic proteotoxic stress when conformationally challenged aggregation-prone proteins are expressed in cancer, metabolic disease, and neurodegenerative disease. The decline in biosynthetic and repair activities that compromises the integrity of the proteome is influenced strongly by genes that control aging, thus linking stress and protein homeostasis with the health and life span of the organism.

/pdf/proteotoxic-stress-and-inducible-chaperone-networks-in-2duicirogo.pdf

Proteotoxic stress and inducible chaperone networks in neurodegenerative disease and aging

The RNA polymerase II (Pol II) enzyme transcribes all protein-coding and most non-coding RNA genes and is globally regulated by Mediator - a large, conformationally flexible protein complex with a variable subunit composition (for example, a four-subunit cyclin-dependent kinase 8 module can reversibly associate with it) These biochemical characteristics are fundamentally important for Mediator's ability to control various processes that are important for transcription, including the organization of chromatin architecture and the regulation of Pol II pre-initiation, initiation, re-initiation, pausing and elongation Although Mediator exists in all eukaryotes, a variety of Mediator functions seem to be specific to metazoans, which is indicative of more diverse regulatory requirements

/pdf/the-mediator-complex-a-central-integrator-of-transcription-1o725u7fkx.pdf

The Mediator complex: a central integrator of transcription

The Mediator is an evolutionarily conserved, multiprotein complex that is a key regulator of protein-coding genes. In metazoan cells, multiple pathways that are responsible for homeostasis, cell growth and differentiation converge on the Mediator through transcriptional activators and repressors that target one or more of the almost 30 subunits of this complex. Besides interacting directly with RNA polymerase II, Mediator has multiple functions and can interact with and coordinate the action of numerous other co-activators and co-repressors, including those acting at the level of chromatin. These interactions ultimately allow the Mediator to deliver outputs that range from maximal activation of genes to modulation of basal transcription to long-term epigenetic silencing.

/pdf/the-metazoan-mediator-co-activator-complex-as-an-integrative-1m7bgovf39.pdf

The metazoan Mediator co-activator complex as an integrative hub for transcriptional regulation

Mediator and the mechanism of transcriptional activation.

Mediator, not holoenzyme, is directly recruited to the heat shock promoter by HSF upon heat shock.

Precise regulation of gene expression is fundamentally required for a broad spectrum of developmental processes in multicellular organisms. Although distinct sequence-specific transcription factors are primarily responsible for this regulation, transcriptional coactivator-corepressor proteins also add a significant secondary layer to the regulation of gene expression. A number of coactivator complexes have been identified in eukaryotes, and their functions in gene activation at specific promoters have been analyzed, primarily in vitro. However, the mechanism by which these transcriptional coactivators regulate gene expression in living organisms is not well understood.

Among eukaryotic transcriptional coactivators, two classes of proteins, Mediator proteins and TATA-binding protein (TBP)-associated factors (TAFs), are central to the process of transcriptional regulation. These proteins were isolated as multiprotein complexes composed of more than 10 polypeptides and associate with the basal transcription machinery (RNA polymerase II [Pol II] and TBP, respectively). Both complexes interact with transcriptional activators and are required for transcriptional activation in reconstituted in vitro systems (3, 8, 40). These facts suggest that Mediator and TAF complexes function as coactivators by relaying transcriptional activation signals from DNA-bound activators to the basal transcription machinery. However, it has not been clearly determined whether Mediator and TAF complexes contribute redundantly or distinctly to the transcriptional activation process, nor have their mechanistic roles in Pol II transcription been clearly deciphered.

Although both Mediator and TAF complexes were initially identified from in vitro assays, recent genetic analyses in yeast suggest that TAFs do not function as general coactivators under physiological conditions. First, the depletion of various TFIID-specific TAFs does not have a significant effect on transcriptional activation of most genes in yeast (12, 28, 41). Second, yeast TAFII145/130 was shown to function as a core promoter selectivity factor rather than a general coactivator (36). These observations are in good agreement with earlier reports that certain TAFs, especially Drosophila TAFII40 (dTAFII40), dTAFII60, dTAFII150, and dTAFII250, directly recognize core promoter elements (7, 39) and thus are likely to act as promoter selectivity factors.

The Mediator complex was first identified in yeast as a multiprotein complex (15, 16) containing functionally distinct modular subassemblies composed of subunits with similar genetic properties (19). Inactivation of individual Mediator proteins causes widely variable effects on yeast gene expression, ranging from deregulation of transcription of a small subset of genes to a genomewide transcriptional defect (12). A number of coactivator complexes related to yeast Mediator have been subsequently isolated in mammals (5, 9, 13, 29, 31, 33, 37). Like yeast Mediator, mammalian complexes play a key role in regulating Pol II transcription in vitro. However, their compositional and functional heterogeneity indicates that there has been a considerable increase in the functional diversity of Mediator complexes during evolution.

In this study, we analyzed the function of the Drosophila Mediator (dMediator) complex in order to gain insight into the mechanism by which these coactivator complexes govern eukaryotic transcriptional activation during Drosophila development. As the first step toward this goal, we isolated a multiprotein complex containing Drosophila homologs of yeast and mammalian Mediator proteins. Our data shows that dMediator interacts physically with several sequence-specific transcription factors and basal transcription machinery and is critically required for transcriptional activation in response to diverse transcriptional activators in vitro. By contrast, the TAF complex is dispensable for transcription of TATA-containing promoters in the presence of dMediator but is required for transcription of TATA-less promoters that depend on an initiator element (Inr) and downstream promoter element (DPE). Our results suggest that dMediator functions as an essential coactivator complex that integrates diverse gene-specific regulatory signals at specific promoters, while TAF complex has its distinct role as a promoter selectivity factor required for the expression of genes with a specific type of core promoter.

/pdf/drosophila-mediator-complex-is-broadly-utilized-by-diverse-1krr3qx38a.pdf

Drosophila Mediator complex is broadly utilized by diverse gene-specific transcription factors at different types of core promoters.

Transcriptional activators interact with diverse proteins and recruit transcriptional machinery to the activated promoter. Recruitment of the Mediator complex by transcriptional activators is usually the key step in transcriptional activation. However, it is unclear how Mediator recognizes different types of activator proteins. To systematically identify the subunits responsible for the signal- and activator-specific functions of Mediator in Drosophila melanogaster, each Mediator subunit was depleted by RNA interference, and its effect on transcriptional activation of endogenous as well as synthetic promoters was examined. The depletion of some Mediator gene products caused general transcriptional defects, whereas depletion of others caused defects specifically related to activation. In particular, MED16 and MED23 were required for lipopolysaccharide- and heat-shock-specific gene expression, respectively, and their activator-specific functions appeared to result from interaction with specific activators. The corequirement of MED16 for other forms of differentiation-inducing factor-induced transcription was confirmed by microarray analysis of differentiation-inducing factor (DIF)- and MED16-depleted cells individually. These results suggest that distinct Mediator subunits interact with specific activators to coordinate and transfer activator-specific signals to the transcriptional machinery.

https://www.pnas.org/content/pnas/101/33/12153.full.pdf

MED16 and MED23 of Mediator are coactivators of lipopolysaccharide- and heat-shock-induced transcriptional activators

The Mediator complex is the major multiprotein transcriptional coactivator complex in Drosophila melanogaster. Mediator components interact with diverse sets of transcriptional activator proteins to elicit the sophisticated regulation of gene expression. The distinct phenotypes associated with certain mutations in some of the Mediator genes and the specific in vitro interactions of Mediator gene products with transcriptional activator proteins suggest the presence of activator-specific binding subunits within the Mediator complex. However, the physiological relevance of these selective in vitro interactions has not been addressed. Therefore, we analyzed dTRAP80, one of the putative activator-binding subunits of the Mediator, for specificity of binding to a number of natural transcriptional activators from Drosophila. Among the group of activator proteins that requires the Mediator complex for transcriptional activation, only a subset of these proteins interacted with dTRAP80 in vitro and only these dTRAP80-interacting activators were defective for activation under dTRAP80-deficient in vivo conditions. In particular, activation of Drosophila antimicrobial peptide drosomycin gene expression by the NF-kappa B-like transcription factor Dif during induction of the Toll signaling pathway was dependent on the dTRAP80 module. These results, and the indirect support from the dTRAP80 artificial recruitment assay, indicate that dTRAP80 serves as a genuine activator-binding target responsible for a distinct group of activators.

Jung Mo Kim

Papers

Mediator, not holoenzyme, is directly recruited to the heat shock promoter by HSF upon heat shock.

Drosophila Mediator complex is broadly utilized by diverse gene-specific transcription factors at different types of core promoters.

MED16 and MED23 of Mediator are coactivators of lipopolysaccharide- and heat-shock-induced transcriptional activators

Signal-induced transcriptional activation by Dif requires the dTRAP80 mediator module.