Transcriptional regulation of small HSP—HSF1 and beyond

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Abstract

The members of the small heat shock protein (sHSP) family are molecular chaperones that play major roles in development, stress responses, and diseases, and have been envisioned as targets for therapy, particularly in cancer. The molecular mechanisms that regulate their transcription, in normal, stress, or pathological conditions, are characterized by extreme complexity and subtlety. Although historically linked to the heat shock transcription factors (HSFs), the stress-induced or developmental expression of the diverse members, including HSPB1/Hsp27/Hsp25, αA-crystallin/HSPB4, and αB-crystallin/HSPB5, relies on the combinatory effects of many transcription factors. Coupled with remarkably different cis-element architectures in the sHsp regulatory regions, they confer to each member its developmental expression or stress-inducibility. For example, multiple regulatory pathways coordinate the spatio-temporal expression of mouse αA-, αB-crystallin, and Hsp25 genes during lens development, through the action of master genes, like the large Maf family proteins and Pax6, but also HSF4. The inducibility of Hsp27 and αB-crystallin transcription by various stresses is exerted by HSF-dependent mechanisms, by which concomitant induction of Hsp27 and αB-crystallin expression is observed. In contrast, HSF-independent pathways can lead to αB-crystallin expression, but not to Hsp27 induction. Not surprisingly, deregulation of the expression of sHSP is associated with various pathologies, including cancer, neurodegenerative, or cardiac diseases. However, many questions remain to be addressed, and further elucidation of the developmental mechanisms of sHsp gene transcription might help to unravel the tissue- and stage-specific functions of this fascinating class of proteins, which might prove to be crucial for future therapeutic strategies.

This article is part of a Directed Issue entitled: Small HSPs in physiology and pathology.

Introduction

Small heat shock protein (sHsp) family is characterized by the presence of an α-crystallin domain. The study of sHsp gene expression contributed in many ways to major breakthroughs in the understanding of the mechanisms of gene transcription, at a time where the concepts of transcription factors and of specific DNA-binding sites were only emerging. These discoveries have concerned the induction of these genes in response to stress, but also their striking expression in the development of diverse organisms from yeast to mammals.

Together with hsp83 and hsp70 genes, the cloning of the Drosophila sHsp genes (hsp22, hsp23, hsp26, hsp27 genes; Holmgren et al., 1981, Riddihough and Pelham, 1986) led to functional studies by transfection of cells or transformation of flies with hybrid genes, containing portions of the regulatory regions of the Hsp genes and reporter genes (Lis et al., 1981, Lis et al., 1983, Pauli et al., 1986, Pelham, 1982). In addition to a TATA box, these approaches contributed to the identification of the heat shock element (HSE; Fig. 1A), a very conserved regulatory sequence present in the 400 bp upstream Hsp genes, that is necessary and sufficient for their inducibility by heat shock, even in heterologous systems. First described as 5′CTnGAAnnTTCnAG3′ (Pelham, 1982), HSE was further refined as an inverted tandem repeats of the motif 5′nTTCnnGAAn3′ (Xiao and Lis, 1988), and more recently through genome-wide and bioinformatical analyses (Trinklein et al., 2004; Fig. 1B; Genomatix, http://www.genomatix.de). The symmetry of this inverted repeat was suggestive of the binding of a regulatory protein, and the following years were an exciting period dedicated to the purification and identification of the transcription factor specifically binding HSE, heat shock factor (HSF; reviewed by Wu, 1995). The presence of multiple elements in the Drosophila hsp70 gene promoter, 2 of which are necessary, and act in a synergistic manner, suggested cooperativity for HSF binding. In parallel, the study of the sHsp gene regulation brought new insights in the regulation of stress-induced chromatin rearrangements upon stress and during development.

  • (1)

    The case of Drosophila small hsp demonstrated the requirement of more than two HSEs for induction by stress, in relation with the more distal positions of HSEs from the TATA box (Bienz and Pelham, 1987). This brought the notion that long-distance effects of HSE require additional transcription factor binding sites, like SP1 (GC-box binding factor) (Bienz and Pelham, 1987).

  • (2)

    Drosophila hsp26 presented an interesting model of chromatin organization that illustrated the notion of DNA-loop formation: the DNA segment, which separates the two HSE-containing hypersensitive sites, is wrapped around a nucleosome, which brings the two HSEs in close vicinity, allowing cooperative interactions between HSFs (Elgin, 1988 in agreement with Wu's results on hsp70 and hsp83, reviewed in Wu, 1995).

  • (3)

    Drosophila hsp26, as dHsp70, is subjected to pausing of the RNA polymerase II, which is already engaged in non-stress conditions on the promoter of Hsp genes (Gilmour and Lis, 1986, Rasmussen and Lis, 1993, Rougvie and Lis, 1988).

  • (4)

    The investigation of sHsp gene expression gave the first best documented evidence for a HSE-independent regulation during development: ovarian induction and late third instar larval/prepupal sHsp expression was found to be regulated by ecdysone via elements distinct from HSEs (Cheney and Shearn, 1983, Cohen and Meselson, 1985, Hoffman and Corces, 1986, Lindquist, 1986, Michaud et al., 1997, Michaud and Tanguay, 2003, Riddihough and Pelham, 1986, Sirotkin and Davidson, 1982, Zimmerman et al., 1983).

In this review, we will describe the major recent findings about the transcriptional regulation of sHsp genes in development, stress, and diseases and will mainly focus on mammalian models (see Table 1). Unless otherwise stated, due to the poorly documented data on many sHsp gene regulation, like HSPB3 and HSPB7, we will mainly focus on HSPB1 and HSPB5.

Heat shock factors form a family of 4 well-described transcription factors (at least) in mammals (Akerfelt et al., 2010) which were named according to the first discovery of their activation by heat shock. Thanks to the universality and robustness of their response to HS, the stress-dependent activation of HSF became a ‘paradigm’: HSF triggers the expression of genes encoding heat shock proteins (Hsps) that function as molecular chaperones, contributing to establish a cytoprotective state to various proteotoxic stress and in several pathological conditions. Although it was believed for a few decades that the role of HSF was entirely dedicated to this protective mechanism, increasing evidence has indicated that this ancient transcriptional program acts genome-widely and performs unexpected functions in the absence of experimentally defined stress. The protective role of HSF has a double-edge sword effect in two types of pathologies that plague the aging population: HSF1 has a major role in longevity and a protective role in neurodegenerative disorders, but in sharp contrast, it promotes cancer, by enabling cells to adapt to the initial oncogenic stress and drastic alterations in energy production, signal transduction, and protein metabolism. In mammals, HSF1 is the major indispensable stress-responsive factor, which is present at a latent state, in the normal cell, and undergoes trimerization, nuclear translocation, and acquires DNA-binding and transcriptional ability upon stress. Its DNA-binding and transcriptional activities are modulated by a series of posttranslational modifications, which also participate to the attenuation of these activities. Part of the action of HSF1 is to release RNA polymerase II, which is already engaged by pausing on the sHsp, as well as the Hsp70 promoters (Rasmussen and Lis, 1993, Rougvie and Lis, 1988). HSF2 modulates the HSF1-dependent induction of Hsp genes upon classical heat shock, likely through the formation of heterotrimers, and was shown to be prominent in the case of mild heat shock at febrile range temperatures (Shinkawa et al., 2011). HSF4 and the newly identified mouse HSF3 do not induce Hsp genes, but regulate non-classical Hsp genes (Fujimoto and Nakai, 2010, Fujimoto et al., 2008). HSF1, 2, and 4 are also essential for embryonic development (reviewed in Abane and Mezger, 2010) and we will see that they participate to the developmental, stress-induced, and disease-related expression of sHsp.

Interestingly, in Drosophila, all hsp promoters contain GA dinucleotide repeats (i.e. (GA or CT)nn; Gilmour et al., 1989) that bind a GAGA factor protein, which keep the hsp promoters “preset” in an active chromatin configuration (Lis and Wu, 1993, Wallrath et al., 1994), ready for the transcriptional activation following a stress. These GAGA factors permit the efficient pausing of the RNA polymerase, which is transcriptionally engaged on the hsp70 and hsp26 promoter (Core and Lis, 2008, Gilmour, 2009, Price, 2008, Rasmussen and Lis, 1993). In the mouse zygote, the onset of embryonic genome transcription requires a GAGA box and GAGA box-binding factors on the Hsp70 promoter (Bevilacqua et al., 2000), but the role of GAGA factors in nucleosome displacement like in Drosophila remains elusive in mammals.

However, analysis of the genome-wide distribution of Drosophila HSF has revealed strong correlation between occupancy of HSEs by HSF and active chromatin marks, suggesting that an active chromatin environment might be necessary for the binding of HSEs by HSF (Guertin and Lis, 2010). In mammals, large-scale analysis of in vivo HSF4 binding sites in lens development and after heat shock also unraveled that HSE occupancy by HSF4 is associated with active chromatin marks (Fujimoto et al., 2008).

HSF2 has an epigenetic role, strictly speaking, in the heritability of a decondensed chromatin status on the Hsp70 gene during mitosis, in a process called “bookmarking” (Xing et al., 2005). This mechanism is supposed to maintain Hsp genes in a transcription competent state in order to allow the quick and robust activation of the gene by HSF1, in the case of a stress in early G1 phase. This bookmarking process also seems to operate on the Hsp27 gene (Wilkerson et al., 2007).

Interestingly, HSF1 has been shown to favor the recruitment of Brg1-containing the SWI/SNF chromatin remodeler via its direct interaction to the Hsp70 promoter (Sullivan et al., 2001). In addition, HSF4β was also shown to recruit Brg1-containing chromatin remodeling complexes to the Hsp27 promoter, as well as for Hsp70, as cells enter the G1 phase of the cell cycle (Tu et al., 2006). A similar scenario could be considered within the mouse αB-crystallin promoter since a highly conserved HSE consensus site is located in the Brg1 regulatory region ([−95/−21]; Fig. 3). Liu et al. have shown that the human chromatin remodeling SWI/SNF-like Brg or hBgm-Associated Factor (BAF) complexes regulate a subset of genes including the αB-crystallin/HSPB5 gene (Liu et al., 2001). This complex by modifying the chromatin structure facilitates different processes including the recruitment of transcription factors to specific target sites (Narlikar et al., 2002). A region immediately upstream the transcription initiation site that is located at the edge of a nucleosome, mediates the Brg1-induced activation of the αB-crystallin gene in transfected cells (Duncan and Zhao, 2007). Furthermore, an AT-rich sequence within this region is bound, in vivo and in vitro, by the non-histone chromatin protein, high mobility group AT-hook 1 (HMGA1) allowing a maximal activation of the αB-crystallin promoter by the direct binding of Brg1 to its Brg1 response element. Notably, this Brg1 response element comprises an activating protein 1 (AP1) binding site (−71 TGACATCA−64) and mutation of this site impairs the activation of Brg1. Since AP1 seems to bind the human αB-crystallin promoter in vivo, it is likely that the binding of AP1 in association with HMGA1 participates to the recruitment of Brg1 to the αB-crystallin promoter (Duncan and Zhao, 2007).

In addition, HSF1 interacts with the transcriptional corepressor CoREST, an integral component of a histone-deacetylase complex that includes also histone lysine-specific demethylase LSD1, during the attenuation phase of the heat shock response, and regulates Hsp70 basal and heat-induced expression levels (Gomez et al., 2008). HSF1 and HSF2 are also able to target histone modifications specific for the stress or stimulus (Akerfelt et al., 2008, Fritah et al., 2009, Fujimoto et al., 2008, Thomson et al., 2004) and HSF2 contributes to chromatin condensation during spermatogenesis (Akerfelt et al., 2008).

The interactions of HSF with various components of the machinery of chromatin remodeling therefore provide a diversity of possible regulations of the sHsp gene family upon stress, and in development and disease.

Section snippets

Small HSPs under developmental and tissue-specific transcriptional regulation

In eukaryotes, as described above, the promoter of many sHsp genes is characterized by the presence of HSEs, which allow their activation under stressful conditions (except, for example, αA-crystallin/HSPB4 promoter). Several other functional regulatory DNA motifs have been reported that contribute to the tissue-specificity and the spatio-temporal pattern of sHSP, particularly during development, in normal conditions.

We will first focus on the regulation of sHsp transcription in the developing

Transcriptional regulation of sHsp expression upon various stresses

Studies of the inducibility of sHsp genes (i.e. HspB1/B5) by various stresses unraveled different modes of transcriptional regulation, which vary according to the cell systems considered: these mechanisms are either dependent on HSF1 – alone or in association with other transcription factors – or independent on HSF1 (i.e. involving regulatory regions distinct of HSE).

Conclusion

The study of sHsp gene transcription in stress, development, and disease has brought many insights on the combinatory action of control elements and transcription factors that operate in a stage- and tissue-dependent manner. HSF-dependent and independent mechanisms have been unraveled that might, in addition, couple the action of transcription factors with associated complexes involved in chromatin remodeling. However, new partners and network interactions remain to be discovered. Using

Acknowledgments

U866 (C. Garrido group) was supported by La Ligue contre le cancer, the Conseil Regional de Bourgogne, the European Commission Seventh Framework Programme (SPEDOC 248835) and the Institut National du Cancer (PAIR). CG group has the label from «La Ligue Contre le Cancer». VM group has been funded by Agence Nationale pour la Recherche (Programme Neurosciences, Neurologie and Psychiatrie), ATC Alcool Inserm, Association pour la Recherche sur le Cancer (ARC, 3609 and 3997), and IREB (Institut de

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    This article is part of a Directed Issue entitled: Small HSPs in physiology and pathology.

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