GAS41 Is Required for Repression of the p53 Tumor Suppressor Pathway during Normal Cellular Proliferation

Abstract

GAS41 is a common subunit of the TIP60 and SRCAP complexes and is essential for cell growth and viability. Here, we report that GAS41 is required for repression of the p53 tumor suppressor pathway during normal cellular proliferation. Either GAS41 small interfering RNA-mediated knockdown of GAS41 expression or specific interruptions of the carboxy-terminal coiled-coil motif of the GAS41 protein activate the p53 tumor suppressor pathway, as evidenced by p53 up-regulation, p53 serine-15 phosphorylation, and p21 transcriptional activation. Activation of the p53 pathway does not result from changes in TIP60 complex assembly or TIP60 coactivator functions for p53, since a TIP60 complex containing a coiled-coil mutant of GAS41 retains the same composition and histone acetyltransferase activity as its wild-type counterpart and since mutant GAS41 does not compromise ectopic p53-dependent transcriptional activation in a reporter gene assay. Finally, we demonstrate that GAS41 is prebound to the promoters of two p53 tumor suppressor pathway genes (p21 and p14 ARF ) in normal unstressed cells but is dissociated from both promoters in response to stress signals that activate p53. Our data suggest that GAS41 plays a role in repressing the p53 tumor suppressor pathway during the normal cell cycle by a TIP60-independent mechanism.Epigenetic mechanisms are now widely accepted as important regulators of gene expression (9, 19). Eucaryotic DNA is packaged into a chromatin structure that consists of arrays of core histone-containing nucleosomes organized, along with linker histone H1, into higher-order structures. Tightly packed nucleosomes generate an inherently repressive structure for gene expression by restricting access of various transcription factors and the general transcription machinery. Moreover, DNA modifications, such as CpG island methylation, and histone tail modifications, such as acetylation, methylation, phosphorylation, and ubiquitylation, allow complex epigenetic controls of nuclear activities that include DNA replication, transcription, DNA repair, and recombination (41).The increasingly large group of chromatin-modifying complexes contains enzymatic activities that covalently modify histone tails. The enzymatic subunits of complexes with histone acetyltransferase (HAT) activity include the H3 HAT GCN5 in Saccharomyces cerevisiae SAGA and human STAGA complexes and the H4 HAT Esa1 in the yeast NuA4 complex (1,22,35). In humans, a NuA4-like complex with TIP60 as the H4 HAT has been described, although there is also a similar complex that lacks HAT activity but shares the TRRAP, p400, Epc1, TIP49, TIP48, BAF53, and ␤-actin components (21, 24). It is not clear whether the last complex has a distinct function or whether it represents an intermediate in the assembly of the TIP60-containing complex. The TIP60 complex was proposed to play a role in DNA repair and apoptosis, and its largest subunit, TRRAP, has been implicated in p53-dependent mdm2 transcriptional activation (3,24,27). TIP60 also has been proposed as an ...

The MYST family of histone acetyltransferases and their intimate links to cancer

“…Amplified copy numbers of the GAS41 (glioma-amplified sequence) gene have been observed in glioblastoma multiforme and astrocytoma (Fischer et al, 1997). More recently, GAS41 has been implicated in suppression of the p53 tumour suppressor pathway during normal cell growth (Park and Roeder, 2006). Like Tip60 itself, the NuA4 subunits BAF53a, TRRAP and p400 have been implicated in c-Myc oncogene-mediated cellular transformation (Fuchs et al, 2001;Nikiforov et al, 2002;Park et al, 2002).…”

The SANT Domain of p400 ATPase Represses Acetyltransferase Activity and Coactivator Function of TIP60 in Basal p21 Gene Expression

“…For the purification of p400 complexes from HeLa cells that stably express Flag-p400 and HA-TIP60, nuclear extracts were prepared by a modified Dignam procedure and directly applied onto a Sepharose CL6B gel filtration column to remove unincorporated free HA-TIP60 (23). Fractions corresponding to the p400 protein peak, as determined by immunoblotting, were combined and subjected to M2 agarose affinity purification.…”

“…Chromatin immunoprecipitation (ChIP) assays were conducted as described previously (23). Briefly, 10 7 cells were cross-linked by the direct addition of formaldehyde (final concentration, 1%) to cells for 10 min at room temperature.…”

The GAS41-PP2Cβ Complex Dephosphorylates p53 at Serine 366 and Regulates Its Stability

“…Because YEATS domain-containing proteins are found in chromatin-modifying and transcription regulatory complexes, this domain is proposed to function in histone and chromatin interactions (18). We previously reported that GAS41 plays a role in repressing the p53 tumor suppressor and appears to reside in a different complex that is independent of the GAS41-containing TIP60 and SRCAP chromatin-remodeling/modifying complexes (19). Here we report the identification of a novel GAS41-PP2C␤ complex that shows phosphatase specificity for phosphorylated serine 366 of p53.…”

“…Protein Purification-For purification of GAS41-PP2C␤ complexes from 293T cells that stably express FLAG-GAS41, nuclear extracts were prepared by a modified Dignam procedure and directly applied to a Sepharose CL6B gel filtration column to remove GAS41-containing nuclear chromatin-remodeling complexes (19). The fractions corresponding to the GAS41 protein peak around 158 kDa of apparent molecular mass were combined and subjected to M2 agarose affinity purification.…”