Benette Phillips

Heat Shock-Induced Interactions of Heat Shock Transcription Factor and the Human hsp70 Promoter Examined by In Vivo Footprinting

Genomic footprinting of the human hsp70 promoter reveals that heat shock induces a rapid binding of a factor, presumably heat shock transcription factor, to a region encompassing five contiguous NGAAN sequences, three perfect and two imperfect matches to the consensus sequence. Arrays of inverted NGAAN sequences have been defined as the heat shock element. No protein is bound to the heat shock element prior to or after recovery from heat shock. Heat shock does not perturb the binding of factors to other regulatory elements in the promoter which contribute to basal expression of the hsp70 gene.

Characterization of constitutive HSF2 DNA-binding activity in mouse embryonal carcinoma cells.

Two distinct murine heat shock transcription factors, HSF1 and HSF2, have been identified. HSF1 mediates the transcriptional activation of heat shock genes in response to environmental stress, while the function of HSF2 is not understood. Both factors can bind to heat shock elements (HSEs) but are maintained in a non-DNA-binding state under normal growth conditions. Mouse embryonal carcinoma (EC) cells are the only mammalian cells known to exhibit HSE-binding activity, as determined by gel shift assays, even when maintained at normal physiological temperatures. We demonstrate here that the constitutive HSE-binding activity present in F9 and PCC4.aza.R1 EC cells, as well as a similar activity found to be present in mouse embryonic stem cells, is composed predominantly of HSF2. HSF2 in F9 EC cells is trimerized and is present at higher levels than in a variety of nonembryonal cell lines, suggesting a correlation of these properties with constitutive HSE-binding activity. Surprisingly, transcription run-on assays suggest that HSF2 in unstressed EC cells does not stimulate transcription of two putative target genes, hsp70 and hsp86. Genomic footprinting analysis indicates that HSF2 is not bound in vivo to the HSE of the hsp70 promoter in unstressed F9 EC cells, although HSF2 is present in the nucleus and the promoter is accessible to other transcription factors and to HSF1 following heat shock. Thus trimerization and nuclear localization of HSF2 do not appear to be sufficient for in vivo binding of HSF2 to the HSE of the hsp70 promoter in unstressed F9 EC cells.

Methylation-associated Transcriptional Silencing of the Major Histocompatibility Complex-linked hsp70 Genes in Mouse Cell Lines

The MHC-linked hsp70 locus consists of duplicated genes, hsp70.1 and hsp70.3, which in primary mouse embryo cells are highly heat shock-inducible. Several mouse cell lines in which hsp70 expression is not activated by heat shock have been described previously, but the basis for the deficiency has not been identified. In this study, genomic footprinting analysis has identified a common basis for the deficient response of the hsp70.1 gene to heat shock in four such cell lines, viz., the promoter is inaccessible to transcription factors, including heat shock transcription factor. Southern blot analyses reveal extensive CpG methylation of a 1.2-kilobase region spanning the hsp70.1 transcription start site and hypermethylation of the adjacent hsp70.3 gene, which is presumably also inaccessible to regulatory factors. Of four additional, randomly chosen mouse cell lines, three show no or minimal hsp70.3 heat shock responsiveness and CpG methylation of both hsp70 genes, and two of the three lines exhibit a suboptimal hsp70.1 response to heat shock as well. In all three lines, the accessibility of the hsp70.1 promoter to transcription factors is detectable but clearly diminished (relative to that in primary mouse cells). Our results suggest that the tandem hsp70 genes are concomitantly methylated and transcriptionally repressed with high frequency in cultured mouse cells.

Hsp70 and Hsc70 are preferentially expressed in differentiated epithelial cells in normal human endometrium and ectocervix.

Two highly related 70K heat shock proteins, encoded by the hsc70 and hsp70 genes, are located in the nucleocytoplasmic compartment of mammalian cells. In contrast to recent cell lines, which express Hsp70 only when stressed, many human cell lines constitutively express Hsp70. The degree to which this reflects constitutive expression of Hsp70 in normal human tissues has not been extensively examined. In this study, we show by immunoblotting that human Hsp70 is constitutively expressed in the ovary, cervix, and endometrium and, by immunohistochemical analysis using Hsp70- and Hsc70-specific antibodies, that Hsp70 and Hsc70 are expressed in distinctive and predominantly overlapping patterns in the cervix and endometrium. In these two tissues, the highest levels of both proteins are seen in differentiated, non-proliferating epithelial cells, which is surprising in light of previous studies suggesting growth stimulation of hsp70 gene expression. These observations suggest the possibility that in certain human tissues, basal expression of the hsp70 and hsc70 genes is co-regulated.

Transcriptional regulation of human hsp70 genes: relationship between cell growth, differentiation, virus infection, and the stress response.

Our understanding of the regulation of the 70-kD family of heat shock genes during human development is necessarily reliant on information obtained from animal studies and from human cell lines which can be induced to differentiate. It has been reported that expression of certain members of the mouse 70-kD heat shock family is regulated during embryogenesis (Bensaude et al. 1983; Bensaude and Morange 1983; Kothary et al. 1987; see Chap. 10) and during spermatogenesis (Krawczyk et al. 1987a; Zakeri and Wolgemuth 1987; Allen et al. 1988; Zakeri et al. 1988; see Chap. 9). Such studies suggest that expression of human heat-shock genes is developmentally regulated. One can also argue, based on our current understanding of the function of this family of genes, that the 70-kD heat shock proteins must play an important role during development and that their expression must, of necessity, be tightly regulated during this process. Heat shock proteins appear to participate in the assembly-disassembly of macromolecular complexes (Georgopoulos and Ang 1990), in transport of polypeptides into organelles such as mitochondria (Chirico et al. 1988; Deshaies et al. 1988; Kang et al. 1990), in trafficking of polypeptides through the endoplasmic reticulum (Dorner et al. 1987), in vesicular uncoating (Ungewickell 1985), in the protection of newly synthesized proteins (Beckmann et al. 1990), and in protein degradation (Chiang et al. 1989). All of these processes must be critical to the successful execution of developmental programs, during which cells alter their patterns of protein synthesis and secretion respond to chemical and hormonal mediators, undergo morphological changes, migrate and interact with other cells, and undergo changes in their growth state. Considering also, as will be detailed in this chapter, the unique ability of the human hsp70 genes to show altered expression in response to environmental stimuli, it seems quite likely that the expression of these genes is highly regulated during development.

In vivo and in vitro Studies on the Activation and Binding of Human Heat-shock Transcription Factor

The rapid transcriptional induction of heat shock genes upon physiological stress such as heat shock is mediated by heat shock transcription factor (HSF) which recognizes a target sequence, the heat shock element (HSE) (Goldenberg et al., 1988; Parker and Topol, 1984; Pelham, 1982; Wu et al., 1987). Heat shock elements consist of an array of inverted repeats of the sequence NGAAN, although the arrangement and number of these NGAAN units can vary (Amin et al., 1988, Xhiao and Lis, 1988). The dynamics of HSF-HSE interactions differ among organisms. In Drosophila, HSF binds to HSE only upon heat shock (Thomas and Elgin, 1988; Wu, 1984), while in yeast, HSF is constitutively bound to HSE (Jakobson and Pelham, 1988; McDaniel et al., 1989; Sorger etal., 1987) and its transcriptional activity is induced by heat shock (Sorger and Pelham, 1988).