Active repression and E2F inhibition by pRB are biochemically distinguishable - PubMed (original) (raw)

Active repression and E2F inhibition by pRB are biochemically distinguishable

J F Ross et al. Genes Dev. 2001.

Abstract

To understand mechanistically how pRB represses transcription, we used a reconstituted transcription assay and compared pRB activity on naked versus chromatin templates. Surprisingly, when pRB was directly recruited to a naked template, no transcriptional repression was observed. However, we observed active repression when the same promoter was assembled into chromatin. Histone deacetylases do not appear to play a role in this observed repression. Further experiments showed repression could occur after preinitiation complex assembly, in contrast with pRB inhibition of E2F, suggesting discrete mechanisms by which pRB directly inhibits an activator such as E2F or actively represses proximally bound transcription factors.

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Figures

Figure 1

Figure 1

Gal4-RB directly represses transcription in vivo and retains the ability to inhibit E2F in vitro. (A) Human pRB-deficient C33A cells were transiently cotransfected with the pG5Sp1E4-CAT reporter containing Gal4 and Sp1 binding sites and the indicated plasmids, and CAT assays were performed as described in Materials and Methods. Either no pRB or increasing amounts of pGal4-RB (+, ++, +++, and ++++ indicate 0.25, 1.25, 2.5, and 5 μg of vector, respectively) were included. As controls, full-length pRB (1.25 μg pCMV-RB) and the Gal4 DNA-binding domain (5 μg pSG42Y) also were tested. Representative data from three independent experiments are shown. (B) Silver-stained SDS–polyacrylamide gels of representative purified factors used in the in vitro transcription reactions. (C) In vitro transcription using p(E2F)4B-Gless as a template. E2F, Gal4-RB, and pRB were included as indicated.

Figure 2

Figure 2

Gal4-RB can repress transcription in vitro on a chromatinized but not a naked template, whereas pRB can inhibit E2F similarly on either template. (A) In vitro transcription using pG5Sp1E4-Gless as a template. Transcription was performed using the indicated factors on a naked (lanes 1_–_7) or chromatin (lanes 8_–_14) template. Five nanograms of Sp1, 20 ng of pRB, 5 ng of Gal4, and 5, 12.5, and 25 ng of Gal4-RB were used per reaction as indicated. The chromatin template was assembled using Drosophila S190 extract. The numbers beneath the lanes represent quantitated relative transcription levels. Basal transcription levels for each panel (lanes 1 and 8) were given unit value. Quantitation was performed as described in Materials and Methods. (B) Transcription reactions were performed as in A, except that reactions included the p(E2F)4B-Gless template, E2F-4/DP-1 (12.5 ng of each subunit per reaction), and 10 ng Gal4-RB. Note that transcription using chromatin results in a lower signal than that of a naked template; hence, autoradiograms displaying transcription from chromatin templates typically required five times longer exposure. (C) In vitro chromatin immunoprecipitations indicate that Gal4-RB does not preclude DNA binding by Sp1. Chromatin was reconstituted with pG5Sp1E4-Gless as a template or mock reconstituted without DNA and incubated with Flag-tagged Sp1, Gal4-RB, or both as indicated above the panel. Proteins and DNA were cross-linked and immunoprecipitated with anti-Flag antibodies as described in Materials and Methods. After reversal of cross-links, samples were electrophoresed and Western blotted sequentially with anti-pRB and anti-Flag antibodies (to detect Sp1). Western blotting with antibodies against Gal4-RB and Sp1 also confirmed that each was present in the indicated reactions before immunoprecipitation (input; data not shown).

Figure 3

Figure 3

Gal4-RB represses transcription on chromatin assembled with recombinant factors. (A) SDS–polyacrylamide gels of the purified factors used to assemble chromatin. The gels containing dNAP1 and ACF were Coomassie- and silver-stained, respectively. (B) Ethidium bromide–stained agarose gels of representative micrococcal nuclease assays to test the assembly of chromatin with S190 extract or recombinant ACF. Increasing amounts of micrococcal nuclease are indicated by the upward sloping triangle. (C) In vitro transcription using chromatin assembled with recombinant factors. Amounts of the indicated factors used were as in Fig. 2. Gal4-RB (12.5 ng) was used in lane 5.

Figure 4

Figure 4

Prior assembly of a preinitiation complex (PIC) does not negate active repression of transcription by Gal4-RB. (A) Diagram of the order of addition experiments. In vitro transcription using (B) pG5Sp1E4-Gless and (C) p(E2F)4BGless templates assembled into chromatin with S190 extract. Sp1 (5 ng) and Gal4-RB (12.5 ng) were included in B, and 12.5 ng of E2F-4/DP-1 and 10 ng of Gal4-RB were added in C as indicated. Gal4-RB was added at the indicated times relative to PIC assembly. Relative transcription levels for B are listed under the lane numbers. Quantitation is relative to basal transcription (lane 1), which was set to unit value as described in the legend to Fig. 2.

Figure 5

Figure 5

Addition of the HDAC inhibitor trichostatin A does not alleviate repression by Gal4-RB. In vitro transcription using chromatin assembled with S190 extract (lanes 1_–_6) or dNAP1 and recombinant ACF (lanes 7_–_12). Sp1 (5 ng), Gal4-RB (12.5 ng), and TSA (0.5 μM) were included as indicated.

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