As demonstrated by the preceding descriptions of nuclear HATs, their in vivo and in vitro functions, and their transcription-related substrates, acetylation is intimately involved with transcriptional regulation on many levels. For many years, it had been known that there was a correlation between histone-acetylated chromatin and activated transcription; HAT and FAT activities were also recognized and partially purified from various organisms in the last several decades. However, only within the last 5 years have HAT catalytic proteins been identified at a molecular level. The revolutionary finding that a transcriptional adaptor protein, Gcn5, was actually a nuclear HAT—followed quickly by similar discoveries with the well-known coactivator p300/CBP, TFIID subunit TAFII250, and other coactivators—established histone acetylation as an apparently ubiquitous mechanism in transcription. This has led to an explosion in HAT-related research, and the lists of HAT proteins, complexes, activities, and substrates continue to grow rapidly.
In many ways, recent advances in molecular biology methods, technology, and informatics have been and will be responsible for the identification and functional characterization of transcriptionally important acetyltransferases. For example, development of the in-gel HAT assay (29
) led to the discovery of Tetrahymena
Gcn5, and the now routine use of protein microsequencing made possible the identification of this and various other HATs. Genome determination and the proliferation of sequence databases are another factor in the discovery of HATs; for example, yeast Esa1 and human PCAF and MORF were originally noticed as database sequences with HAT homology. The imminent completion of human genome sequencing should lead to the identification of additional human HATs. It should be noted that some HATs (e.g., TAFII
250 and nuclear receptor coactivators) have no recognizable homology to known acetyltransferase motifs and will not be discovered in this way, but the recognition of histone acetylation as a major regulatory mechanism has led to the now widespread use of HAT assays in the characterization of transcription-related proteins, resulting in perhaps unanticipated findings of certain HAT activities (e.g., TFIIIC). This trend is expected to continue in the future.
As the body of information about transcriptional regulation grows and cellular processes previously considered distinct are found to be intricately linked, HAT functional studies are benefiting from multiple, unified scientific approaches. Biochemical and molecular biology techniques are being used to purify HAT complexes and characterize thoroughly their activities and subunits, genetics are providing information about in vivo function, and structural studies, such as the recent GNAT determinations, are giving insights into mechanisms and interactions. These types of investigations will continue, and two recently developed techniques in particular also show promise for future functional determinations of identified HATs and other proteins. Chromatin immunoprecipitation (ChIP) is a way in which chromatin can be retrieved from cells and analyzed for acetylation state or transcriptional proteins at specific genes, providing a wealth of information about in vivo HAT functions and complexes. For a wider functional view, whole-genome analyses with oligonucleotide microarrays have been and will be used to analyze cells' RNA and assess the impact of HAT mutations on the expression of all genes for a given organism or cell type. This has already been performed for Gcn5 and other interesting transcriptional proteins in S. cerevisiae
). In the future, such expression studies will likely be carried out with other HATs in S. cerevisiae
and also in more complex eukaryotes as the technology and genomic data advance. Thus, in complementary ways, chromatin immunoprecipitation assays and microarrays may provide detailed data about HAT action and other transcriptional regulation under various conditions.
In addition, it should be remembered that histones and regulatory proteins receive a variety of functionally important covalent modifications in vivo, not just acetylation (227
). Although this review has focused rather narrowly on chromatin- and transcription-related acetylation, future studies must increasingly address the interplay of multiple modifications with one another, with other activities such as ATP-dependent chromatin remodeling, and with the mechanisms that reverse or antagonize these processes (e.g., deacetylation and chromatin assembly). Recent investigations have established that there are close relationships among all of these functions, but the nature of these must be better defined by future research. Another relevant issue is that the state of chromatin also influences other significant nuclear processes besides transcription, such as DNA replication, recombination, and repair. For example, a recent study revealed that V(D)J recombination of antibody genes is tightly correlated with histone H3 acetylation (162
); the connection between such processes and various modes of chromatin alteration will require further investigation. Finally, a largely unexplored frontier in this field is the topic of higher-order chromatin structure, whose effects on transcription must be addressed along with those of nucleosomes in future studies, contributing to the eventual goal of a detailed, overall understanding of the regulation of gene expression in the eukaryotic cell.