Turku Centre for Biotechnology

An overview of the mammalian heat shock factor (HSF) family members and their biological functions. HSFs contribute to multiple normal physiological processes and pathologies through direct regulation of their target genes. The HSF target genes that have been identified in vivo are shown. HSF1 was originally recognized as the principal stress-responsive regulator of the heat shock response, but now HSF2 is known to modulate HSF1-mediated expression of heat shock protein (HSP) genes through heterocomplex formation. On heat shock, HSF1 and HSF2 accumulate into nuclear stress bodies (NSBs), where they bind to satellite III repeats. HSF1 is also a regulator of immune responses and cancer. So far, the regulation of HSP genes in ageing has most intensively been examined in Caenorhabditis elegans. Both HSF1 and HSF2 have been ascribed regulatory functions in several developmental processes, such as oogenesis, spermatogenesis and corticogenesis. HSF4 is involved in the development of different sensory organs in cooperation with HSF1, but has no role in the heat shock response. Murine HSF3 is the most recently identified mammalian HSF, which participates in the heat shock response by binding to the PDZ domain-containing 3 (Pdzk3) promoter10 (Åkerfelt et al., 2010).

Our main topic is the molecular mechanisms by which the different members of the HSF family are regulated during normal development and under stressful conditions. In particular, we investigate the expression and activity of HSF1 and HSF2. We have found that HSF1 activity is primarily regulated by various post-translational modifications (PTMs), e.g. acetylation, phosphorylation and sumoylation. All these PTMs are induced by stress stimuli but their effects on HSF1 vary. Upon stress, HSF1 undergoes phosphorylation-dependent sumoylation within a bipartite motif, which we found in many transcriptional regulators and gave name PDSM (phosphorylation-dependent sumoylation motif). Stress-inducible hyperphosphorylation and sumoylation of HSF1 occur very rapidly, whereas acetylation of HSF1 increases gradually, indicating a role for acetylation in the attenuation phase of the HSF1 activity cycle. Among multiple lysine residues targeted by acetylation, K80 is located within the DNA-binding domain of HSF1 and its acetylation is required for reducing HSF1 DNA-binding activity. Importantly, the duration of HSF1 DNA-binding activity can be prolonged or diminished by chemical compounds either activating or inhibiting the activity of the NAD⁺-dependent deacetylase SIRT1. Our current focus is on a complex network of PTMs to decipher the post-translational signature of HSF1.

In contrast to HSF1, which is a stable protein evenly expressed in most tissues and cell types, the amount of HSF2 varies and correlates with its activity. Recently, we reported the first evidence for the ubiquitin E3 ligase APC/C (anaphase-promoting complex/cyclosome) mediating ubiquitination and degradation of HSF2 during the acute phase of the heat shock response. The stress-related composition and role of APC/C are unknown and form our major future goal. We will also determine the stress effects on the cell cycle, adding a new dimension to the research field.

Using mouse spermatogenesis as a model system, we discovered an inverse correlation between the cell- and stage-specific wave-like expression patterns of HSF2 and a specific microRNA, miR-18, which is a member of the Oncomir-1/miR-17~92 cluster. Intriguingly, miR-18 was found to repress the expression of HSF2 by directly targeting its 3’UTR. To investigate the in vivo function of miR-18, we developed a novel method T-GIST (Transfection of Germ cells in Intact Seminiferous Tubules) and showed that inhibition of miR-18 in intact mouse seminiferous tubules leads to increased HSF2 protein levels and altered expression of HSF2 target genes, including the Y-chromosomal multi-copy genes that we previously had reported as novel HSF2 targets in the testis. Our original finding that miR-18 regulates HSF2 activity in spermatogenesis links miR-18 to HSF2-mediated physiological processes and opens a whole new window of opportunities to elucidate the physiological and stress-related functions of HSF2, either alone or in conjunction with HSF1.

We were the first to report that HSF2 forms a complex with HSF1 and regulates the heat shock response. Our studies on HSF1-HSF2 heterotrimers and their impact on various target genes are designed to elucidate the roles of HSFs in protein-misfolding disorders, such as neurodegenerative diseases, as well as in aging and cancer progression. Most studies have focused on HSF1, but it is important to consider the existence of multiple HSFs and interactions between them, especially when searching for potential drugs to modify their expression and/or activity. Our ongoing genome-wide ChIP-sequencing experiments to compare HSF1 and HSF2 occupancy in non-stressed and stressed cells for better understanding their actions in various chromatin environments, including cells arrested in mitosis, should give news insights on these multi-faceted transcriptional regulators.