Transcriptional stimulation by the DNA binding protein Hap46/BAG-1M involves hsp70/hsc70 molecular chaperones

Abstract

The hsp70/hsc70-associating protein Hap46 of human origin, also called BAG-1M (Bcl-2-associated athanogene 1), has been characterized previously as a DNA binding protein, which is able to stimulate transcription. By use of in vitro assays we now show that Hap46-mediated transcriptional activation can occur from linearized as well as from supercoiled circular DNA and does not require the presence of a transcription promoter. Accordingly, we observed no preferential binding of Hap46 to overlapping DNA fragments covering the sequence of the cytomegalovirus (CMV) early promoter, thus suggesting non-specific binding. The C-terminal deletion variant Hap46DeltaC47, which is unable to associate with hsp70/hsc70 molecular chaperones, produced greatly diminished effects on transcription, indicating a significant involvement of hsp70/hsc70 chaperones but not an absolute requirement. In contrast, deletion of the acidic hexarepeat region, as in variant Hap46Delta12-62, did not disturb transcriptional stimulation. While full-length Hap46 readily formed complexes with a series of structurally unrelated transcription factors, variant Hap46DeltaC47 proved incapable of doing so. Together these data suggest that transcriptional stimulation is a major biological activity of Hap46 and point to involvement of hsp70/hsc70 molecular chaperones in transcription in concert with Hap46, thus providing a link between hsp70/hsc70 molecular chaperones and components of the transcription machinery.

Figure 1

Domain structure of Hap46. I, DNA binding region; II, acidic hexarepeat region (consensus sequence Ser-Glu-Glu-X-Thr-Arg); III, ubiquitin-like region. IV, hsp70/hsc70 binding region. Deletion variants are shown schematically in relation to the domain structure of Hap46.

Figure 2

Hap46 stimulates transcription from circular and linearized DNA. (A) In the experiments of lanes 2 and 3, plasmid pcDNA3/CAT was used for transcription assays with HeLa nuclear extracts, as described in Materials and Methods, either in the absence or presence of full-length Hap46, as indicated. In the experiments of lanes 4–9, prokaryotic DNA of 517 bp (lanes 4–6) and eukaryotic DNA of 2.3 kb (lanes 7–9) were similarly used in the absence (lanes 4 and 7) or presence of full-length Hap46 (lanes 5 and 8) or Hap46ΔC47 (lanes 6 and 9), as described in Materials and Methods. The same result was obtained with other prokaryotic or eukaryotic DNAs of 396 and 0.9 kb, respectively (not shown). As control, a 1485 bp template containing the CMV promoter was used with no Hap46 added (lane 1). (B) The same linear CMV promoter-containing template was used in transcription assays either in the absence (lane 1) or presence of full-length Hap46 (lane 2), Hap46Δ12-62 (lane 3) and Hap46ΔC47 (lane 4). Labeled RNA was analyzed by gel electrophoresis and autoradiography. (A) Lanes 7–9 were analyzed on 4% polyacrylamide gels, all others were run on 6% gels. Positions of RNA size markers are shown along margins. Arrowheads mark the position of the CMV promoter-specific transcript of 363 nt.

Figure 3

Hap46 binds to circular DNA. Plasmid pcDNA3/CAT was incubated with full-length Hap46 and hsc70, as indicated, and submitted to electrophoretic moblity-shift assays in 0.8% agarose gels, as described in Materials and Methods. Ethidium stained bands are shown here in dark.

Figure 4

Binding of Hap46 to fragments of the CMV promoter. (A) The CMV promoter sequence of 655 bp was divided by PCR into six portions of ∼130 bp each and five portions of ∼235 bp, as detailed in Materials and Methods. (B) The above DNA fragments were incubated with Hap46 and analyzed by gel electrophoresis, as described in Materials and Methods. Ethidium stained bands are shown in dark.

Figure 5

Interaction of Hap46 with transcription factors. Fusion proteins of GST with full-length Hap46 (lanes 2) or Hap46ΔC47 (lanes 3) were attached to GSH–Sepharose and used as affinity matrices for in vitro expressed radiolabeled transcription factors, as described in Materials and Methods. For controls, plain GSH–Sepharose was used (lanes 1). Rabbit reticulocyte lysate was used as such in the experiment of (A). Eluates were analyzed by SDS–PAGE followed either by autoradiographic detection of radiolabeled bands or, in the case of hsc70 (A), by specific immunoblotting.

Figure 6

Interaction of Hap46 and hsc70 with the estrogen receptor. The estrogen receptor attached to a specific immunomatrix was incubated with the GST–Hap46 fusion protein, as described in Materials and Methods. The experiment of lane 2 contained minimal amounts of endogenous hsc70 co-purified with the receptor, while further hsc70 (5 µg) was added in the experiment of lane 3. As control (lane 1), the plain immunomatrix was used without estrogen receptor but with hsc70 (5 µg) added.