p300 and PCAF act cooperatively to mediate transcriptional activation from chromatin templates by notch intracellular domains in vitro - PubMed (original) (raw)

p300 and PCAF act cooperatively to mediate transcriptional activation from chromatin templates by notch intracellular domains in vitro

Annika E Wallberg et al. Mol Cell Biol. 2002 Nov.

Abstract

Ligand activation of Notch receptors leads to release of the intracellular receptor domain (Notch IC), which translocates to the nucleus and interacts with the DNA-binding protein RBP-Jkappa to control expression of specific target genes. A number of proteins have been shown to interact with Notch ICs and to modulate target gene activation, but the precise function of and interplay between these factors is not known. This report investigates the Notch IC-interacting proteins, p300, PCAF, and Mastermind-like 1 (MAML1), in an in vitro transcription system with purified factors and naked DNA or chromatin templates. MAML1, RBP-Jkappa, and Notch IC are all required for optimal transcription from DNA, whereas transcription from chromatin requires, in addition, p300, which interacts with MAML1. The transcriptional activity of p300 requires acetyl coenzyme A, indicating that it functions as a histone acetyltransferase when mediating Notch IC function. PCAF is unable to promote transcription on its own but enhances Notch IC-mediated transcription from chromatin in conjunction with p300. These data define a critical role for p300 in the potentiation of Notch IC function by MAML1 and PCAF, provide the first evidence for cooperativity between PCAF and p300 in Notch IC function, and also indicate direct effects of RBP-Jkappa, Notch IC, and MAML1 on the general transcription machinery.

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Figures

FIG. 1.

FIG. 1.

MAML1 stimulates Notch IC-driven transcription from a DNA template. (A) Diagram showing the 12RBP-Jκ template. (B) Schematic representation of the in vitro transcription assays indicating the order in which the reagents were added. (C) Coomassie blue staining of purified FLAG-tagged proteins used in the transcription assays. (D) The DNA template was transcribed following RBP-Jκ and Notch1 IC or Notch3 IC binding in the presence or absence of MAML1, as indicated.

FIG. 2.

FIG. 2.

Notch function on a chromatin template is dependent on both MAML1 and p300. (A) DNA supercoiling assay for chromatin assembly. Chromatin was assembled on relaxed circular plasmids, and the resulting superhelicity was checked on a 1% agarose gel which was visualized by ethidium bromide staining. Lanes contain CsCl purified supercoiled (sc) plasmid (lane 1), topoisomerase 1-relaxed (rel) plasmid used for assembly (lane 2), and plasmid purified after chromatin assembly with S190 extract (lane 3). (B) Micrococcal nuclease (MNase) analysis of assembled chromatin on a 1.2% agarose gel. A 123-bp DNA ladder was used as size marker (M). (C) A Coomassie blue-stained SDS-PAGE gel shows the purified p300 and PCAF proteins used in the transcription assays. (D) Transcription from chromatin (lanes 1 to 9) or DNA (lanes 10 and 11) templates. The chromatin templates were incubated with RBP-Jk, Notch1 IC or Notch3 IC, MAML1, and p300 as indicated. Acetyl-CoA was subsequently added to all lanes.

FIG. 3.

FIG. 3.

p300-mediated transcription is dependent on acetyl-CoA. (A) In vitro transcription of chromatin templates assembled with S190 extract. (B) In vitro transcription of chromatin templates assembled with the recombinant ACF system. (C) DNA supercoiling assay for chromatin assembly. Lane 1 shows CsCl-purified supercoiled (sc) plasmid, lane 2 shows topoisomerase 1-relaxed (rel) plasmid used for assembly, and lane 3 shows plasmid purified after chromatin assembly with the recombinant ACF system. (D) Micrococcal nuclease (MNase) analysis of chromatin assembled with the recombinant ACF system. A 123-bp DNA ladder was used as size marker (M). (E) Coomassie blue-stained SDS-PAGE gels show the purified HeLa histones, Acf-1, ISWI, and NAP-1 proteins used for assembly.

FIG. 3.

FIG. 3.

p300-mediated transcription is dependent on acetyl-CoA. (A) In vitro transcription of chromatin templates assembled with S190 extract. (B) In vitro transcription of chromatin templates assembled with the recombinant ACF system. (C) DNA supercoiling assay for chromatin assembly. Lane 1 shows CsCl-purified supercoiled (sc) plasmid, lane 2 shows topoisomerase 1-relaxed (rel) plasmid used for assembly, and lane 3 shows plasmid purified after chromatin assembly with the recombinant ACF system. (D) Micrococcal nuclease (MNase) analysis of chromatin assembled with the recombinant ACF system. A 123-bp DNA ladder was used as size marker (M). (E) Coomassie blue-stained SDS-PAGE gels show the purified HeLa histones, Acf-1, ISWI, and NAP-1 proteins used for assembly.

FIG. 3.

FIG. 3.

p300-mediated transcription is dependent on acetyl-CoA. (A) In vitro transcription of chromatin templates assembled with S190 extract. (B) In vitro transcription of chromatin templates assembled with the recombinant ACF system. (C) DNA supercoiling assay for chromatin assembly. Lane 1 shows CsCl-purified supercoiled (sc) plasmid, lane 2 shows topoisomerase 1-relaxed (rel) plasmid used for assembly, and lane 3 shows plasmid purified after chromatin assembly with the recombinant ACF system. (D) Micrococcal nuclease (MNase) analysis of chromatin assembled with the recombinant ACF system. A 123-bp DNA ladder was used as size marker (M). (E) Coomassie blue-stained SDS-PAGE gels show the purified HeLa histones, Acf-1, ISWI, and NAP-1 proteins used for assembly.

FIG. 3.

FIG. 3.

p300-mediated transcription is dependent on acetyl-CoA. (A) In vitro transcription of chromatin templates assembled with S190 extract. (B) In vitro transcription of chromatin templates assembled with the recombinant ACF system. (C) DNA supercoiling assay for chromatin assembly. Lane 1 shows CsCl-purified supercoiled (sc) plasmid, lane 2 shows topoisomerase 1-relaxed (rel) plasmid used for assembly, and lane 3 shows plasmid purified after chromatin assembly with the recombinant ACF system. (D) Micrococcal nuclease (MNase) analysis of chromatin assembled with the recombinant ACF system. A 123-bp DNA ladder was used as size marker (M). (E) Coomassie blue-stained SDS-PAGE gels show the purified HeLa histones, Acf-1, ISWI, and NAP-1 proteins used for assembly.

FIG. 3.

FIG. 3.

p300-mediated transcription is dependent on acetyl-CoA. (A) In vitro transcription of chromatin templates assembled with S190 extract. (B) In vitro transcription of chromatin templates assembled with the recombinant ACF system. (C) DNA supercoiling assay for chromatin assembly. Lane 1 shows CsCl-purified supercoiled (sc) plasmid, lane 2 shows topoisomerase 1-relaxed (rel) plasmid used for assembly, and lane 3 shows plasmid purified after chromatin assembly with the recombinant ACF system. (D) Micrococcal nuclease (MNase) analysis of chromatin assembled with the recombinant ACF system. A 123-bp DNA ladder was used as size marker (M). (E) Coomassie blue-stained SDS-PAGE gels show the purified HeLa histones, Acf-1, ISWI, and NAP-1 proteins used for assembly.

FIG. 4.

FIG. 4.

Binding of p300 to MAML1, Notch1 IC, and Notch3 IC. FLAG-tagged proteins bound to M2-agarose were tested for interaction with purified p300 in an in vitro protein interaction assay. p300 was detected in a Western blot by using an antibody (Santa Cruz Biotechnology) against the N-terminal domain of p300. The input represents 10% of the p300 protein used in each binding reaction. The negative control is M2-agarose without any bound FLAG-tagged protein.

FIG. 5.

FIG. 5.

PCAF acts synergistically with p300 to mediate Notch IC activity from chromatin templates. Transcription assay with chromatin templates assembled with S190 extract. Acetyl-CoA, RBP-Jκ, and MAML1 were added to all reactions, and Notch1 IC, Notch3 IC, p300, and PCAF were added as indicated.

FIG. 6.

FIG. 6.

Notch function on naked DNA is not affected by p300 and PCAF. The DNA templates were transcribed following RBP-Jκ, Notch1 IC or Notch3 IC, and MAML1 binding in the presence or absence of p300 and PCAF as indicated (lanes 5 to 12). p300 and PCAF do not significantly influence basal transcription (lanes 1 to 4).

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