Abstract
Analysis of the heat shock element (HSE)-binding proteins in extracts of rodent cells, during heat shock and their post-heat shock recovery, indicates that the regulation of heat shock response involves a constitutive HSE-binding factor (CHBF), in addition to the heat-inducible heat shock factor HSF1. We purified the CHBF to apparent homogeneity from HeLa cells using column chromatographic techniques including an HSE oligonucleotide affinity column. The purified CHBF consists of two polypeptides with apparent molecular masses of 70 and 86 kDa. Immunoblot and gel mobility shift analysis verify that CHBF is identical or closely related to the Ku autoantigen. The DNA binding characteristics of CHBF to double-stranded or single-stranded DNA are similar to that of Ku autoantigen. In gel mobility shift analysis using purified CHBF and recombinant human HSF1, CHBF competes with HSF1 for the binding of DNA sequences containing HSEs in vitro. Furthermore, when Rat-1 cells were co-transfected with human Ku expression vectors and the hsp70-promoter-driven luciferase reporter gene, thermal induction of luciferase is significantly suppressed relative to cells transfected with only the hsp70-luciferase construct. These data suggest a role of CHBF (or Ku protein) in the regulation of heat response in vivo.
Highlights
Analysis of the heat shock element (HSE)-binding proteins in extracts of rodent cells, during heat shock and their post-heat shock recovery, indicates that the regulation of heat shock response involves a constitutive HSE-binding factor (CHBF), in addition to the heat-inducible heat shock factor HSFI
Th ere is a tight temporal corr elati on betw een the disappea rance of HSFI-HSE binding activity and th e recovery of CHBF-HSE binding activity in Rat-L, Chinese ham ster HA-l (Fig. lB ), a nd HeLa cells
Mammalian heat shock transcription factor HSFI has been the target of extensive studies relating to heat shock gene activation in recent years
Summary
Analysis of the heat shock element (HSE)-binding proteins in extracts of rodent cells, during heat shock and their post-heat shock recovery, indicates that the regulation of heat shock response involves a constitutive HSE-binding factor (CHBF), in addition to the heat-inducible heat shock factor HSFI. In gel mobility shift analysis using purified CHBF and recombinant human HSFl, CHBF competes with HSFI for the binding of DNA sequences containing HSEs in vitro. When Rat-I cells were co-transfected with human Ku expression vectors and the hsp70-promoter-driven luciferase reporter gene, thermal induction of luciferase is significantly suppressed relative to cells transfected with only the hsp70-luciferase construct These data suggest a role of CHBF (or Ku protein) in the regulation of heat response in vivo. Unstressed cells contain a constitutive HSE binding activity that appears as a faster migrating complex, while a distinct HSE binding activity (HSFl), evidenced as a slower migrating complex, is present only in the heatshocked cells, Our recent studies on the response of rodent cells to heat shock, sodium arsenite, or sodium salicylate indicated that a high level of HSFI-HSE binding activity by itself is neither sufficient nor necessary for the induction of hsp mRNA transcription [11]. The tight temporal inverse correlation between HSE-binding ability of HSFI and HSE-binding ability of CHBF suggests that this correlation may be functionally significant, The molecular and biochemical basis of this observation is unclear, We have previously suggested that the inverse correlation between the HSE binding activity of HSFI and CHBF may reflect the involvement of both in the regulation of heat shock gene expression, the former as a positive and the latter as a negative regulator
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