Chromatin is not an inert structure, but rather an instructive DNA scaffold that can respond to external cues to regulate the many uses of DNA. A principle component of chromatin that plays a key role in this regulation is the modification of histones. There is an ever-growing list of these modifications and the complexity of their action is only just beginning to be understood. However, it is clear that histone modifications play fundamental roles in most biological processes that are involved in the manipulation and expression of DNA. Here, we describe the known histone modifications, define where they are found genomically and discuss some of their functional consequences, concentrating mostly on transcription where the majority of characterisation has taken place.

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Regulation of chromatin by histone modifications

Activation of p53 Sequence-Specific DNA Binding by Acetylation of the p53 C-Terminal Domain

All eukaryotic cells have an extensive membranous labyrinth network of branching tubules and flattened sacs called the endoplasmic reticulum (ER). Approximately one-third of all cellular proteins are translocated into the lumen of the ER where post-translational modification, folding, and oligomerization occurs. The ER provides a unique oxidizing compartment for the folding of membrane and secretory proteins that are destined to the cell surface, as well as for proteins destined to other intracellular organelles, such as lysosomes and the Golgi compartment. Numerous cellular proteins reside within the ER through a mechanism that requires their continuous vesicle-mediated retrieval from post-ER compartments within the early secretory pathway. These ERresident proteins are chaperones and catalysts of protein folding that form a matrix on which newly synthesized proteins attain their final conformation. The ER is also the site of synthesis of cellular lipids and sterols. In addition, the ER is the major signal-transducing organelle within the cell that continuously responds to environmental cues to release calcium. The ER is exquisitely sensitive to alterations in homeostasis, where, upon a variety of different stimuli, signals are transduced from the ER to the cytoplasm and the nucleus to eventually result in adaptation for survival or induction of apoptosis. The immediate response occurs at the translational apparatus, whereas changes in gene expression promote long-term adaptation or apoptotic cell death. Recent evidence supports findings that these signaling pathways influence pathogenesis associated with viral infection and genetic disease. The purpose of this review is to summarize what is presently known about the diversity of molecular signaling mechanisms that coordinate the complex ER stress response at the translational and transcriptional level in yeast and in higher eukaryotic cells.

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Stress signaling from the lumen of the endoplasmic reticulum: coordination of gene transcriptional and translational controls

Nuclear receptors (NR) comprise a family of transcription factors that regulate gene expression in a liganddependent manner. Members of the NR superfamily include receptors for steroid hormones, such as estrogens (ER) and glucocorticoids (GR), receptors for nonsteroidal ligands, such as thyroid hormones (TR) and retinoic acid (RAR), as well as receptors that bind diverse products of lipid metabolism, such as fatty acids and prostaglandins (for review, see Beato et al. 1995; Chambon 1995; Mangelsdorf and Evans 1995). The NR superfamily also includes a large number of so-called orphan receptors for which regulatory ligands have not been identified (Mangelsdorf and Evans 1995). Although many orphan receptors are likely to be regulated by small-molecular-weight ligands, other mechanisms of regulation, such as phosphorylation (Hammer et al. 1999; Tremblay et al. 1999) have also proven to be of importance. Remarkably, the sequence of the Caenorhabditis elegans genome has revealed the presence of >200 members of the NR family, suggesting a critical role of these proteins in environmental adaptation (Sluder et al. 1999). Although mammalian genomes are unlikely to contain such a large complement of these factors, >24 distinct classes of NR have been identified in humans, and these factors exert diverse roles in the regulation of growth, development, and homeostasis. Based on their importance in biology and medicine, as well as the relatively simple mechanism of regulation, NR represent one of the most intensively studied and best-understood classes of transcription factors at the molecular level. Members of the NR family regulate transcription by several mechanisms (Fig. 1). Nuclear receptors can activate or repress target genes by binding directly to DNA response elements as homoor heterodimers or by binding to other classes of DNA-bound transcription factors. A subset of NRs, including TR and RAR, can actively repress target genes in the presence or absence of ligand binding, and many NR have been demonstrated to inhibit transcription in a ligand-dependent manner by antagonizing the transcriptional activities of other classes of transcription factors. These activities have been linked to interactions with general classes of molecules that appear to serve coactivator or corepressor function. In this review, we will discuss recent progress concerning the molecular mechanisms by which NR cofactor interactions serve to activate or repress transcription.

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The coregulator exchange in transcriptional functions of nuclear receptors

More than 30 years ago, Vincent Allfrey proposed that histone acetylation was associated with transcriptional activity in eukaryotic cells (Allfrey et al. 1964; Pogo et al. 1966). Subsequently, acetylated core histones were shown to preferentially associate with transcriptionally active chromatin (Sealy and Chalkley 1978; Vidali et al. 1978; Hebbes et al. 1988). Acetylation occurs at lysine residues on the amino-terminal tails of the histones, thereby neutralizing the positive charge of the histone tails and decreasing their affinity for DNA (Hong et al. 1993). As a consequence, histone acetylation alters nucleosomal conformation (Norton et al. 1989), which can increase the accessibility of transcriptional regulatory proteins to chromatin templates (Lee et al. 1993; Vettese-Dadey et al. 1996). Taken together, these observations suggested how histone acetylation could result in increased transcriptional activity in vivo. However, there was essentially no information about the cause and effect relationship between histone acetylation and transcriptional activity or about the underlying molecular mechanisms. A mechanistic and physiologically relevant connection between histone acetylation and transcriptional regulation was initially provided by two independent lines of evidence. First, yeast cells unable to acetylate the histone H4 tail because of mutations of the target lysine residues show altered patterns of transcription (Durrin et al. 1991). However, these mutations broadly affect chromatin structure in vivo, and hence are likely to influence other molecular processes involving DNA (e.g., DNA replication and repair, recombination, chromosome segregation). Second, treatment of mammalian cells with potent inhibitors of histone deacetylase activity such as trapoxin or trichostatin A resulted in increased expression of a variety of genes (Yoshida et al. 1995). However, these drugs might inhibit other cellular targets, and they affect a variety of cellular processes, including cell proliferation, apoptosis, differentiation, and DNA synthesis. Although these observations were suggestive, understanding of the relationship between chromatin structure and transcription regulation was hampered significantly by a lack of knowledge about the enzymes that acetylate and deacetylate histones. In the past 2 years, our understanding of the causal relationship between histone acetylation and gene expression has been enhanced dramatically by the identification of proteins with intrinsic histone acetylase and deacetylase activity (Brownell et al. 1996; for recent reviews, see Grunstein 1997; Pazin and Kadonaga 1997; Wade et al. 1997). Of particular significance, some of these enzymes had been identified previously as components of the RNA polymerase II (Pol II) transcription machinery itself, proteins that associate with transcriptional regulatory factors, or proteins that positively or negatively affect transcription in vivo. These discoveries have led to a major paradigm shift. It is now clear that chromatin structure and modification can not be viewed as a process that is independent of transcriptional initiation, that is, chromatin is not simply a structure that serves to compact DNA in the nucleus and provide a relatively passive substrate for the action of transcription factors. Instead, histone acetylases and deacetylases provide a critical link between chromatin structure and transcriptional output, and this link is now accessible to experimental intervention. This review will focus on molecular mechanisms by which histone acetylation affects transcriptional activity in living cells.

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Histone acetylation and transcriptional regulatory mechanisms

The transcriptional adaptor protein Gcn5 has been identified as a nuclear histone acetyltransferase (HAT). Although recombinant yeast Gcn5 efficiently acetylates free histones, it fails to acetylate histones contained in nucleosomes, indicating that additional components are required for acetylation of chromosomal histones. We report here that Gcn5 functions as a catalytic subunit in two high-molecular-mass native HAT complexes, with apparent molecular masses of 0.8 and 1.8 megadalton (MD), respectively, which acetylate nucleosomal histones. Both the 0.8- and 1.8-MD Gcn5-containing complexes cofractionate with Ada2 and are lost in gcn5delta, ada2delta, or ada3delta yeast strains, illustrating that these HAT complexes are bona fide native Ada-transcriptional adaptor complexes. Importantly, the 1.8-MD adaptor/HAT complex also contains Spt gene products that are linked to TATA-binding protein (TBP) function. This complex is lost in spt20/ada5delta and spt7delta strains and Spt3, Spt7, Spt20/Ada5, Ada2, and Gcn5 all copurify with this nucleosomal HAT complex. Therefore, the 1.8-MD adaptor/HAT complex illustrates an interaction between Ada and Spt gene products and confirms the existence of a complex containing the TBP group of Spt proteins as demonstrated by genetic and biochemical studies. We have named this novel transcription regulatory complex SAGA (Spt-Ada-Gcn5-Acetyltransferase). The function of Gcn5 as a histone acetyltransferase within the Ada and SAGA adaptor complexes indicates the importance of histone acetylation during steps in transcription activation mediated by interactions with transcription activators and general transcription factors (i.e., TBP).

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Yeast Gcn5 functions in two multisubunit complexes to acetylate nucleosomal histones: characterization of an Ada complex and the SAGA (Spt/Ada) complex.

Core histones isolated from normal and butyrate-treated HeLa cells have been reconstituted into nucleosome cores in order to analyze the role of histone acetylation in enhancing transcription factor binding to recognition sites in nucleosomal DNA. Moderate stimulation of nucleosome binding was observed for the basic helix-loop-helix factor USF and the Zn cluster DNA binding domain factor GAL4-AH using heterogeneously acetylated histones. However, by coupling novel immunoblotting techniques to a gel retardation assay, we observed that nucleosome cores containing the most highly acetylated forms of histone H4 have the highest affinity for these two transcription factors. Western analysis of gel-purified USF-nucleosome and GAL4-AH-nucleosome complexes demonstrated the predominant presence of acetylated histone H4 relative to acetylated histone H3. Immunoprecipitation of USF-nucleosome complexes with anti-USF antibodies also demonstrated that these complexes were enriched preferentially in acetylated histone H4. These data show that USF and GAL4-AH preferentially interact with nucleosome cores containing highly acetylated histone H4. Acetylation of histone H4 thus appears to play a primary role in the structural changes that mediate enhanced binding of transcription factors to their recognition sites within nucleosomes.

C D Allis

Papers

Yeast Gcn5 functions in two multisubunit complexes to acetylate nucleosomal histones: characterization of an Ada complex and the SAGA (Spt/Ada) complex.

Acetylation of histone H4 plays a primary role in enhancing transcription factor binding to nucleosomal DNA in vitro.