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. 2001 Jul 1;15(13):1619-24.
doi: 10.1101/gad.900101.

The ATM-related Domain of TRRAP Is Required for Histone Acetyltransferase Recruitment and Myc-dependent Oncogenesis

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Free PMC article

The ATM-related Domain of TRRAP Is Required for Histone Acetyltransferase Recruitment and Myc-dependent Oncogenesis

J Park et al. Genes Dev. .
Free PMC article

Abstract

The ATM-related TRRAP protein is a component of several different histone acetyltransferase (HAT) complexes but lacks the kinase activity characteristic of other ATM family members. We identified a novel function for this evolutionarily conserved domain in its requirement for the assembly of a functional HAT complex. Ectopic expression of TRRAP protein with a mutation in the ATM-related domain inhibits Myc-mediated oncogenic transformation. The Myc-binding region of TRRAP maps to a separable domain, and ectopic expression of this domain inhibits cell growth. These findings demonstrate that the ATM-related domain of TRRAP forms a structural core for the assembly and recruitment of HAT complexes by transcriptional activators.

Figures

Figure 1
Figure 1
The ATM/PI-3 kinase domain of TRRAP is critical for the recruitment of histone acetyltransferase activity. (A) Schematic representation of wild-type, full-length TRRAP (top) with the ∼300 C-terminal amino acids homologous to the ATM/PI-3 kinase family members indicated as ATM. Below is shown a series of TRRAP internal fragments, C-terminal truncation mutants, and internal deletion mutants with their respective sizes at the right. (B) ATM-domain dependence for HAT recruitment. Complexes containing transiently expressed FLAG-tagged TRRAP or TRRAP mutants were isolated from HEK293 cells (McMahon et al. 1998). The FLAG–TRRAP or TRRAP mutants were eluted from beads and assayed for HAT activity on calf thymus core histones. Reaction mixtures were resolved on an SDS-PAGE gradient gel (4%–15%) and transferred to a nitrocellulose membrane. The upper section was probed with anti-FLAG and anti-hGCN5 antibodies. Cell lysate containing 100 μg of protein was included as a positive control for hGCN5 (lane left of lane 1). The bottom portion of the membrane below the 30-kD molecular mass marker was stained with Ponceau-S and subjected to fluorography for HAT activities. The 97-kD band common to lanes 17 in the hGCN5 panel is a nonspecific band that arises from the anti-FLAG beads, but is not found with cell lysates (lane left of lane 1).
Figure 2
Figure 2
Expression of a TRRAP mutant defective in HAT recruitment inhibits c-Myc oncogenic activity. (A) Primary rat embryo fibroblasts were transfected with expression vectors for c-Myc, H-RasG12V, wild-type full-length TRRAP, and the HAT-defective TRRAP(1–3760) truncation mutant in the different combinations indicated. (y axis) Number of transformed foci. Three plates were assayed for each bar. (B) TRRAP and TRRAP mutants do not inhibit colony growth. Primary rat embryo fibroblasts were transfected as indicated along with pSG5 puromycinR plasmid. PuromycinR colonies were counted after 14 d. Two plates were assayed for each bar in the graph.
Figure 3
Figure 3
The ATM/PI-3 kinase domain of TRRAP is critical for interaction with hSPT3. HEK293 cells were transiently transfected with expression vectors for FLAG-tagged TRRAP fragment or its deletion mutant, TRRAP(1–3713). Cell lysates were prepared and immunoprecipitated with anti-FLAG antibodies. Each precipitate was resolved by SDS-PAGE and analyzed for the extent of its coprecipitation with endogenous hSPT3. To check the relative expression levels of TRRAP fragments and hGCN5 recruitment, the same membrane was reprobed with anti-FLAG antibodies (top) and anti-hGCN5 (middle).
Figure 4
Figure 4
TRRAP amino acids 1899–2026 mediate Myc binding. (A) In vivo coimmunoprecipitation. HEK293 cells were transiently cotransfected with CMV-driven expression vectors for c-Myc and FLAG-tagged full-length TRRAP or a series of TRRAP C-terminal truncation mutants. Cell lysates were prepared and the FLAG-tagged TRRAP protein or mutants were immunoprecipitated with anti-FLAG antibodies. Precipitates were resolved by SDS-PAGE and analyzed by Western blotting for anti-c-Myc antibodies (bottom). The same membrane was also probed with anti-FLAG antibodies to detect TRRAP expression (top). Apparent molecular masses in kilodaltons of protein markers are indicated at the left of fluorogram. (B) Localization of the c-Myc-binding domain. HEK 293 cells were transiently transfected with expression vectors for FLAG-tagged TRRAP fragment. Cell lysates were prepared and immunoprecipitated with anti-FLAG antibodies. Each precipitate was resolved by SDS-PAGE and analyzed for the extent of its coprecipitation with endogenous c-Myc (top). Cell lysate from the c-Myc transfection was included as a positive control (lane left of lane 1). To check the relative expression levels of TRRAP fragments, the same membrane was reprobed with anti-FLAG antibodies (bottom). (C) An internal region of TRRAP (spanning amino acids 1591–2026) interacts with N-Myc. GST fusion proteins containing the indicated TRRAP regions were expressed in E. coli and bound to glutathione beads. Equal amounts of bead-bound fusion proteins were incubated with affinity-purified FLAG-tagged N-Myc protein. After washing, interacting proteins were subjected to SDS-PAGE analysis and immunoblotted with anti-FLAG antibody. The FLAG-N-Myc starting material (20% of input) was run with the bound material.

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