The Identification of GCN5-Related Proteins as Histone Acetyltransferases Links Chromatin Acetylation to Gene Activation

The expression of protein-encoding genes in eukaryotes requires the coordinated activity of DNA-sequence specific transcriptional activators with the assembly of multicomponent transcription complexes and the recruitment of RNA polymerase II. The genetic template itself is organized as a hierarchical complex of DNA wrapped with histones and other proteins to form nucleosomes and higher-order chromatin structures. Gene activation occurs within this chromatin context in vivo and is tightly correlated with histone modifications, most notably the posttranslational acetylation of several highly conserved lysine residues within the amino termini of the core histones.

The relationship between histone acetylation and transcription was first observed more than 30 years ago, prior even to the discovery of the nucleosome. At that time, Allfrey (1) wrote that "modifications of histone structure, acetylation in particular, may affect the capacity of histones to inhibit ribonucleic acid synthesis in vivo [raising] the possibility that minor changes in histone structure offer a means of switching -on or -off RNA synthesis at different loci along chromosomes." Since then, it has been generally accepted that acetylation serves to alter chromatin structure, thereby facilitating transcription factor access to the underlying DNA.

The precise effects of acetylation remain unclear, however, and Allfrey's original hypothesis remains the driving force behind attempts to understand the role of chromatin structure in the regulation of gene expression. The correlation of acetylation and transcription is well established, and numerous studies have shown that acetylation is mediated by the activity of histone acetyltransferase (HAT) and deacetylase (HD) enzyme systems (2). Understanding these enzymes is therefore important for understanding the function and regulation of acetylation.

To begin to approach this problem, I devised a reverse genetics strategy for studying a type-A HAT (a chromatin-bound enzyme suspected to be responsible for transcription-related acetylation). A key to my isolation of HAT A was the development of an SDS-PAGE-based acetyltransferase activity assay that detects polypeptides that possess intrinsic HAT activity from crude or fractionated cellular extracts (3). This assay is extremely useful because it uses SDS-PAGE as an enzyme purification step by using acetyltransferase activity to identify catalytically active HAT polypeptides directly within an SDS gel. Using this assay, I identified, isolated, and eventually cloned the gene for a catalytically active HAT of 55 kDa (dubbed p55) from macronuclei of the ciliated protozoan, Tetrahymena thermophila (4).

The sequence of Tetrahymena p55 clearly identifies it as a GCN5-related protein, a conserved family of proteins to date found in yeast (5), flies (6), and humans (7). Consistent with the high degree of similarity between p55 and other family members, I and others have demonstrated that indeed all known GCN5-related proteins possess intrinsic HAT activity (4, 6, 9). Moreover, analyses of the histones acetylated by yeast Gcn5p reveal that the specific lysine residues acetylated are identical to those shown previously to be enriched in transcriptionally active chromatin in vivo (10). Earlier genetic and biochemical studies in yeast have defined GCN5 along with several other proteins (ADA1, 2, 3, and 5) as components of a transcription adaptor complex required for the full activity of several transcriptional activators (i.e., GCN4), although no biochemical function was known (5). My results suggest a clear biochemical function for GCN5 and related proteins as HATs, providing the first direct link between histone acetylation and factors required for activated gene expression.

Both in vivo and in vitro studies have shown that GCN5 family proteins function as components of multisubunit complexes, thus assembly and disassembly of these complexes may serve to regulate transcription-related HAT activity (11). Experimental evidence for this idea was provided recently by the discovery that the adenoviral oncogene product E1A specifically disrupts the association of a human GCN5-related protein, PCAF (p300/CBP associated factor), with the transcriptional co-activator CBP (CREB-binding protein; (9)). Analogous to the GCN5-ADA complex in yeast, the human PCAF-CBP complex is required to mediate transcription by several transcriptional activators including CREB and c-FOS/JUN (12). E1A-specific targeting and disruption of PCAF-CBP complexes in turn effects the expression of genes under the control of these activators, ultimately leading to altered cell growth and transformation (9). Recently it was reported that CBP itself possesses intrinsic HAT activity (13), adding to the mounting interest in CBP as a multifunctional protein with a central role in transcriptional regulation. Indeed, due to its interactions with several hormone-induced nuclear receptors, CBP is now thought to serve as an 'integrator' of external signaling pathways at the level of transcription-related chromatin remodeling (14).

In addition to GCN5-related proteins and CBP, our laboratory recently used the acetyltransferase activity gel assay to identify intrinsic HAT activity in TAFII250, an component of the basal transcription apparatus (15). Despite the common ability to acetylate histones, however, sequence comparisons have failed to reveal significant homology among these proteins, with one notable exception. Each of these nuclear HATs possesses at least one copy of the bromodomain, a conserved motif found in a restricted set of polypeptides, all of which are known to be components of multisubunit complexes involved in transcriptional activation (16). Interestingly, yHat1p, a cytoplasmic type-B HAT that acetylates newly synthesized histones prior to chromatin assembly (as opposed to transcription), does not possess a bromodomain (17). These observations led to our proposal that the bromodomain may represent a "signature sequence" that distinguishes HAT As from HAT Bs, and more importantly provides a mechanism for directing HAT As (as well as other transcription factors) to genes targeted for activation within the chromatin template (4).

Certainly the specific sequence of events involved in activated gene expression remains incompletely defined. However, recent developments including the identification of HATs as well as HDs (18) have begun to clarify the relationship between factors that modulate chromatin structure and transcription itself. The discovery that several proteins involved in transcriptional activation possess intrinsic acetyltransferase activity not only links acetylation and transcriptional regulation, but also implies that acetylation, like phosphorylation, may be involved in the regulation of a wider variety of cellular processes than previously thought.

References

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