Sharon Y. Roth

Tetrahymena Histone Acetyltransferase A: A Homolog to Yeast Gcn5p Linking Histone Acetylation to Gene Activation

We report the cloning of a transcription-associated histone acetyltransferase type A(HAT A). This Tetrahymena enzyme is strikingly homologous to the yeast protein Gcn5, a putative transcriptional adaptor, and we demonstrate that recombinant Gcn5p possesses HAT activity. Both the ciliate enzyme and Gcn5p contain potential active site residues found in other acetyltransferases and a highly conserved bromodomain. The presence of this domain in nuclear A-type HATs, but not in cytoplasmic B-type HATs, suggests a mechanism whereby HAT A is directed to chromatin to facilitate transcriptional activation. These findings shed light on the biochemical function of the evolutionarily conserved Gcn5p-Ada complex, directly linking histone acetylation to gene activation, and indicate that histone acetylation is a targeted phenomenon.

Essential and redundant functions of histone acetylation revealed by mutation of target lysines and loss of the Gcn5p acetyltransferase

The Gcn5p histone acetyltransferase exhibits a limited substrate specificity in vitro. However, neither the specificity of this enzyme in vivo nor the importance of particular acetylated residues to transcription or cell growth are well defined. To probe these questions, we mutated specific lysines in the N‐termini of histones H3 and H4 and examined the effects of these mutations in yeast strains with and without functional GCN5. We found that in vivo, GCN5 is required either directly or indirectly for the acetylation of several sites in H3 and H4 in addition to those recognized by the recombinant enzyme in vitro. Moreover, in the absence of GCN5, cells accumulate in G2/M indicating that Gcn5p functions are important for normal cell‐cycle progression. Mutation of K14 in H3, which serves as the major target of recombinant Gcn5p acetylation in vitro, confers a strong, synthetic growth defect in gcn5 cells. Synergistic growth defects were also observed in gcn5 cells carrying mutations in lysine pairs (K8/K16 or K5/K12) in histone H4. Strikingly, simultaneous mutation of K14 in H3 and K8 and K16 in H4 to arginine, or deletion of either the H3 or the H4 N‐terminal tail, results in the death of gcn5 cells. Mutation of these same three sites to glutamine is not lethal. Indeed, this combination of mutations largely bypasses the need for GCN5 for transcriptional activation by Gal4–VP16, supporting an important role for histone acetylation in Gcn5p‐mediated regulation of transcription. Our data indicate that acetylation of particular lysines in histones H3 and H4 serves both unique and overlapping functions important for normal cell growth, and that a critical overall level of histone acetylation is essential for cell viability.

Histone acetylation and chromatin assembly: a single escort, multiple dances?

The remarkable flurry in identification of chromatin-modifying activities in recent times has been accompanied by unexpected connections between these activities and cellular transformation. The possible link between Rb function as a tumor suppressor and the function of p48 as a “histone escort” is intriguing, but is at present very speculative. However, disruption of interactions between a Gcn5p (HAT A) homolog, P/CAF, and its cofactors, p300 or CBP, by the product of the viral E1A oncogene is required for E1A-mediated cellular transformation (Yang et al. 1996xYang, X.J, Ogryzko, V.V, Nishikawa, J, Howard, B.H, and Nakatani, Y. Nature. 1996; 382: 319–324CrossRef | PubMed | Scopus (1184)See all ReferencesYang et al. 1996). Translocation of another putative acetylase, MOZ, and in-frame fusion to CBP is associated with specific subtypes of acute myeloid leukemias (Borrow et al. 1996xBorrow, J, Stanton, V.P Jr., Andresen, J.M, Becher, R, Behm, F.G, Chaganti, R.S.K, Civin, C.I, Disteche, C, Dube, I, Frischauf, A.M, Horsman, D, Mitelman, F, Volina, S, Watmore, A.E, and Housman, D.E. Nature Genet. 1996; 13: 33–41CrossRef | Scopus (518)See all ReferencesBorrow et al. 1996). MOZ is homologous to the yeast gene SAS2 (Something About Silencing) which is required for silencing in yeast (Reifsnyder et al. 1996xReifsnyder, C, Lowell, J, Clarke, A, and Pillus, L. Nature Genet. 1996; 13: 42–49CrossRef | Scopus (212)See all ReferencesReifsnyder et al. 1996), suggesting the human gene may also participate in silencing functions. Understanding the nature, regulation, and specificity of these activities is no longer just the pursuit of those interested in understanding the structure and function of chromatin, but is now directly relevant to our understanding of both normal cellular regulatory processes and abnormal processes which lead to oncogenesis.

Loss of Gcn512 leads to increased apoptosis and mesodermal defects during mouse development

Histone acetyltransferases regulate transcription, but little is known about the role of these enzymes in developmental processes. Gcn5 (encoded by Gcn5l2) and Pcaf, mouse histone acetyltransferases, share similar sequences and enzymatic activities. Both interact with p300 and CBP (encoded by Ep300 and Crebbp, respectively), two other histone acetyltransferases that integrate multiple signalling pathways. Pcaf is thought to participate in many of the cellular processes regulated by p300/CBP (refs 2–8), but the functions of Gcn5 are unknown in mammalian cells. Here we show that the gene Pcaf is dispensable in mice. In contrast, Gcn5l2-null embryos die during embryogenesis. These embryos develop normally to 7.5 days post coitum (d.p.c.), but their growth is severely retarded by 8.5 d.p.c. and they fail to form dorsal mesoderm lineages, including chordamesoderm and paraxial mesoderm. Differentiation of extra-embryonic and cardiac mesoderm seems to be unaffected. Loss of the dorsal mesoderm lineages is due to a high incidence of apoptosis in the Gcn5l2 mutants that begins before the onset of morphological abnormality. Embryos null for both Gcn5l2 and Pcaf show even more severe defects, indicating that these histone acetyltransferases have overlapping functions during embryogenesis. Our studies are the first to demonstrate that specific acetyltransferases are required for cell survival and mesoderm formation during mammalian development.

Nucleosomes are positioned with base pair precision adjacent to the alpha 2 operator in Saccharomyces cerevisiae.

Analysis of the chromatin structure of minichromosomes containing the binding site for the yeast alpha 2 repressor protein by indirect end‐labeling has previously indicated that nucleosomes are stably positioned over sequences adjacent to the alpha 2 operator in the presence of the repressor. Development of a primer extension assay for nucleosome position now allows a more detailed examination of the location of these nucleosomes relative to the operator sequence, and indicates that nucleosomes are precisely and stably positioned both translationally and rotationally over sequences adjoining the operator. In addition, this assay enables analysis of the chromatin structure of single copy, genomic sequences. Chromatin structures determined for two genes regulated by alpha 2, STE6 and BAR1, are consistent with nucleosomes precisely positioned downstream of the operator sequence, incorporating promoter elements, in alpha cells but not in a‐cells. The location of these nucleosomes relative to the operator sequence is highly analogous to that observed in the minichromosome. The stability of the nucleosomes adjacent to the operator together with the precision of their location suggests that they may play a role in repression of a specific gene expression by alpha 2. Further, the primer extension assay allows a comparison of the structure of these positioned nucleosomes formed in vivo to that previously described for core particles reconstituted in vitro.

The Growth Suppressor PML Represses Transcription by Functionally and Physically Interacting with Histone Deacetylases

ABSTRACT The growth suppressor promyelocytic leukemia protein (PML) is disrupted by the chromosomal translocation t(15;17) in acute promyelocytic leukemia (APL). PML plays a key role in multiple pathways of apoptosis and regulates cell cycle progression. The present study demonstrates that PML represses transcription by functionally and physically interacting with histone deacetylase (HDAC). Transcriptional repression mediated by PML can be inhibited by trichostatin A, a specific inhibitor of HDAC. PML coimmunoprecipitates a significant level of HDAC activity in several cell lines. PML is associated with HDAC in vivo and directly interacts with HDAC in vitro. The fusion protein PML-RARα encoded by the t(15;17) breakpoint interacts with HDAC poorly. PML interacts with all three isoforms of HDAC through specific domains, and its expression deacetylates histone H3 in vivo. Together, the results of our study show that PML modulates histone deacetylation and that loss of this function in APL alters chromatin remodeling and gene expression. This event may contribute to the development of leukemia.

Mammalian GCN5 and P/CAF Acetyltransferases Have Homologous Amino-Terminal Domains Important for Recognition of Nucleosomal Substrates

ABSTRACT The yeast transcriptional adapter Gcn5p serves as a histone acetyltransferase, directly linking chromatin modification to transcriptional regulation. Two human homologs of Gcn5p have been reported previously, hsGCN5 and hsP/CAF (p300/CREB binding protein [CBP]-associated factor). While hsGCN5 was predicted to be close to the size of the yeast acetyltransferase, hsP/CAF contained an additional 356 amino-terminal residues of unknown function. Surprisingly, we have found that in mouse, both the GCN5and the P/CAF genes encode proteins containing this extended amino-terminal domain. Moreover, while a shorter version of GCN5 might be generated upon alternative or incomplete splicing of a longer transcript, mRNAs encoding the longer protein are much more prevalent in both mouse and human cells, and larger proteins are detected by GCN5-specific antisera in both mouse and human cell extracts. Mouse GCN5 (mmGCN5) andmmP/CAF genes are ubiquitously expressed, but maximum expression levels are found in different, complementary sets of tissues. Both mmP/CAF and mmGCN5 interact with CBP/p300. Interestingly,mmGCN5 maps to chromosome 11 and cosegregates withBRCA1, and mmP/CAF maps to a central region of chromosome 17. As expected, recombinant mmGCN5 and mmP/CAF both exhibit histone acetyltransferase activity in vitro with similar substrate specificities. However, in contrast to yeast Gcn5p and the previously reported shorter form of hsGCN5, mmGCN5 readily acetylates nucleosomal substrates as well as free core histones. Thus, the unique amino-terminal domains of mammalian P/CAF and GCN5 may provide additional functions important to recognition of chromatin substrates and the regulation of gene expression.

Chromatin and transcription

The compaction of DNA into chromatin in the eukaryotic nucleus poses many obstacles to transcription. Individual nucleosomes as well as higher order structures limit access of cis‐acting regulatory elements to trans‐acting factors. The structural nature of this inhibition and the mechanisms by which chromatin is remodeled to facilitate the regulation of gene expression have remained puzzles for many years. Recent advances highlight the intimate and dynamic interplay between transcription proteins and components of chromatin, providing new clues to long‐standing questions. A transcriptional adaptor complex has been discovered to house histone acetylase activity. A chromatin remodeling “machine” has been found to be part of the RNA polymerase II holoenzyme. Identification of new factors that affect the organization of functional chromatin domains in yeast, flies, and mammals provides new insights into the organization of higher order chromatin structures, as well as the nature of boundaries that restrict these domains. These compelling discoveries and others define a new and exciting threshold for our understanding of the many connections between chromatin and transcription.—Edmondson, D. G., Roth, S. Y. Chromatin and transcription. FASEB J. 10, 1173‐1182 (1996)

A Transcriptionally Active tRNA Gene Interferes with Nucleosome Positioning in Vivo

Incorporation into a positioned nucleosome of a cis-acting element essential for replication in Saccharomyces cerevisiae disrupts the function of the element in vivo [R. T. Simpson, Nature (London) 343:387-389, 1990]. Furthermore, nucleosome positioning has been implicated in repression of transcription by RNA polymerase II in yeast cells. We have now asked whether the function of cis-acting elements essential for transcription of a gene transcribed by RNA polymerase III can be similarly affected. A tRNA gene was fused to either of two nucleosome positioning signals such that the predicted nucleosome would incorporate near its center the tRNA start site and essential A-box element. These constructs were then introduced into yeast cells on stably maintained, multicopy plasmids. Competent tRNA genes were transcribed in vivo and were not incorporated into positioned nucleosomes. Mutated, inactive tRNA genes were incorporated into nucleosomes whose positions were as predicted. This finding demonstrates that the transcriptional competence of the tRNA gene determined its ability to override a nucleosome positioning signal in vivo and establishes that a hierarchy exists between cis-acting elements and nucleosome positioning signals.

Unequal access: Regulating V(D)J recombination through chromatin remodeling

Alt and colleagues (Yancopoulos and Alt, 1985), supposes that the packaging of the TCR and Ig loci into chromatin differs in T cells and B cells, and that this packaging varies according to the activity of the loci. In accordance with this hypothesis, active V(D)J recombi-Chromatin is organized into repeat units called nucleo-somes, which are in turn folded into progressively higher order structures. Each nucleosome contains two mole-The ability of the vertebrate immune system to respond cules of the four core histones, H2A, H2B, H3, and H4 to a wide variety of antigens depends on the pro-and 147 bp of DNA spooled around the outside of the grammed rearrangement of genes encoding the antigen histone octamer. A fifth histone, H1, binds to the exterior receptor proteins, the T cell receptors (TCR) and immu-of the nucleosome core and to the linker DNA between noglobulins (Ig), during lymphocyte differentiation. Por-cores. Interactions between core histones or between tions of the receptors that encode the antigen binding linker histones in neighboring nucleosomes facilitate domains are assembled from separately encoded V higher order chromatin packing. (variable), D (diversity), and J (joining) gene segments. Both individual nucleosomes and more folded struc-Biochemical studies have shown that the V(D)J recombi-tures limit access of transacting factors to DNA (see nase machinery is composed of the RAG-1 and RAG-2 Sudarsanam and Winston, 2000; Wade et al., 1997 and proteins plus at least one accessory protein (HMG1 or references therein). These limitations have been exten-HMG2). The recombinase binds to recombination signal sively studied in the context of transcription. Distortion sequences (RSS) immediately adjacent to the V, D, and of the DNA wound around the histone octamer can pre-J coding segments, brings an appropriate pair of DNA clude factor binding, and the histones themselves can segments together to form a synaptic complex, and sterically hinder interactions of the DNA with other pro-then introduces a pair of double-strand breaks (DSB) precisely between each RSS and its adjoining coding segment (Figure 1). The broken ends are then rejoined in a recombinant configuration with the participation of several double-strand break repair factors (Oettinger, 1999). The Accessibility Hypothesis Developing lymphocytes harbor seven complex immune receptor loci (four TCR and three Ig) that are rearranged by a common recombinase. However, TCR genes rearrange only in T cells, and Ig genes rearrange fully only in B cells. In addition, recombination is regulated temporally: specific loci (such as the …