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Protein Complexes That Modify Chromosomes
Summary: Jerry Workman's laboratory has purified and studies several protein complexes that modify regions of chromosomes to regulate the expression of the underlying genes.
Protein Complexes That Modify Chromatin and Regulate Gene Transcription
Eukaryotic chromosomes comprise DNA that is complexed with small basic proteins, histones, and other proteins to generate chromatin, a nucleic acid:protein complex. The tight association of these proteins with DNA provides a level of transcription control and contributes to epigenetic mechanisms of gene regulation. For example, the amino-terminal tail domains of the core histone proteins are sights of numerous post-translational modifications. In addition to potential effects on chromatin structure, these modifications act as binding sites/receptors for protein complexes that activate or repress gene transcription. Thus histone modification can be used to generate a "code" of signals on the surface of the chromosome fiber, which provides regulatory information above that contained in the DNA sequence. Our laboratory is focused on studying the protein complexes that carry out these histone modifications and those that recognize the resulting signals.
Purification and Analysis of Histone Acetyltransferase Complexes
Since the discovery of histone acetylation over three decades ago, a strong connection has been shown between the acetylation of histone proteins and transcriptional activity. Over the past seven years, a number of the enzymes that carry out these modifications have been identified. We have purified and characterized the native protein HAT (histone acetyltransferase) complexes bearing these enzymes to understand their recruitment to promoter DNA and their function in gene regulation. Thus far we have identified and characterized six such complexes from yeast. These include the 2-MDa SAGA (Spt-Ada-Gcn5-acetyltransferase) complex, the related SLIK (SAGA-like) complex, the 0.8-MDa ADA complex, the 1.3-MDa NuA4 (nucleosome acetyltransferase of histone H4) complex, the 0.4-MDa NuA3 complex, and the 0.4-MDa SAS (something about silencing) complex.
Recruitment of Histone Acetyltransferase Complexes by Transcription Activators to Acetylate Promoter Nucleosomes
The SAGA complex comprises more than 20 different subunits, which include several different classes of proteins implicated in transcription regulation. SAGA contains Ada proteins, the TATA box–binding protein (TBP) group of Spt gene products, a subset of TBP-associated factors (TAFIIs), and the 400-kDa Tra1 protein. Tra1 is related to the ataxia telangiectasia mutated (ATM) family of phosphatidylinositol 3-kinases and is the yeast homolog of the human TRRAP protein, which plays an essential role in cellular transformation by the c-Myc and E2F oncogenes. Tra1 is also the largest subunit of the NuA4 complex, which also contains a number of gene products implicated in transcriptional regulation. Its catalytic subunit is Esa1 (essential SAS-related acetyltransferase 1).
Although SAGA primarily acetylates histone H3 and NuA4 histone H4, they share a number of biochemical properties. Our studies have shown that both SAGA and NuA4 interact specifically with the activation domains of promoter-binding transcription activators. When recruited to a promoter through these interactions, either SAGA or NuA4 acetylates promoter-proximal nucleosomes, which results in transcriptional enhancement. Importantly, the interaction of HAT complexes with transcription activators is a step at which gene activation can be regulated. For example, the Gal80 repressor protein specifically blocks the interaction of SAGA and NuA4 with the Gal4 transcription activation domain. This prevents the Gal4 protein from recruiting these HAT complexes to promoters where it is bound. Activation of Gal4-regulated genes therefore involves inactivation of Gal80, leading to recruitment of HAT complexes to Gal4-regulated promoters.
Through a series of biochemical and genetic experiments, we have shown that the interactions of SAGA and NuA4 with transcription activators occurs through the shared Tra1 subunit. Tra1 directs the HAT complexes to promoters via interactions with transcription activators where they lay down a narrow (SAGA) or wide (NuA4) patch of acetylation on nucleosomal histones surrounding the promoter. In addition to characterizing the SAGA and NuA4 complexes further, we are also investigating the function of the acetylated patches laid down by these HATs at promoters in chromatin.
Promoter Recruitment and Retention of the SWI/SNF Chromatin Remodeling Complex: Linking Histone Acetylation to Nucleosome Disruption
The yeast SWI/SNF complex is a 2-Mda protein complex that is required for the transcription of a subset of yeast genes. Mechanistic studies, by others and us, have demonstrated that SWI/SNF uses the energy of ATP hydrolysis to alter the structure and location of nucleosomes. Through a series of biochemical experiments we have demonstrated that the SWI/SNF complex is also recruited to promoters by direct interactions with the same promoter-binding transcription activators that recruit the SAGA and NuA4 complexes. This suggests that these complexes act in concert during chromatin remodeling, which is consistent with genetic interactions between SAGA and SWI/SNF subunits. We have found that after its recruitment by an activator, the retention of SWI/SNF on the promoter can persist following loss of the activator if adjacent nucleosomes contain acetylated histones. Indeed, prior recruitment of the SAGA or NuA4 HAT complexes to a promoter stabilizes the subsequent binding of SWI/SNF by acetylating promoter-proximal nucleosomes.
The Swi2/Snf2 subunit of the SWI/SNF complex contains a bromodomain, which has been proposed to be an acetyl-lysine–binding domain. By purifying and analyzing a SWI/SNF complex from which this domain has been deleted, we have found that it is required for SWI/SNF retention on acetylated promoter nucleosomes. Bromodomains are also found in a number of protein complexes involved in transcriptional regulation, including the general transcription factor TFIID and the RSC chromatin-remodeling complex. Thus histone acetylation has the potential to provide high-affinity interaction sites for bromodomain-containing complexes in chromatin. The SAGA complex itself contains two bromodomains, in the Gcn5 and Spt7 subunits, and is able to stabilize its own binding to promoter nucleosomes by acetylation of histones.
Histone Acetyltransferase Complexes That Function in Transcription Elongation, DNA Replication, and Gene Silencing
In contrast to the SAGA and NuA4 complexes, the ADA, NuA3, and SAS complexes do not interact with transcription activators. The ADA complex contains a subset of the proteins found in SAGA (including Gcn5 as the catalytic subunit) and additional unique subunits. The NuA3 complex contains the Sas3 protein as its catalytic subunit. Two hybrid interaction studies revealed that the Sas3 protein is able to interact with the Spt16 protein. Spt16 is part of an abundant protein complex known as the CP or FACT (facilitates chromatin transcription) complex, which has been implicated in DNA replication and transcription elongation. Biochemical studies revealed that both the NuA3 and ADA complexes interact directly with the amino-terminal region of Spt16. This suggests that these two HAT complexes may play overlapping roles in the function of Spt16. Consistent with this is the fact that double deletions of SAS3 and AHC1 (a unique subunit of the ADA complex) recapitulate phenotypes associated with DNA replication defects seen in Spt16 amino-terminal truncations. Moreover, these same Spt16 truncations lead to reduced levels of global histone H3 acetylation. Our current model is that the ADA and NuA3 complexes contribute to widespread/global histone acetylation via their interactions with Spt16. Global acetylation is in turn important for the efficiency of DNA replication and transcription elongation.
Sas2 is a putative acetyltransferase with significant homology to SAS3 and ESA1. We purified the protein complex containing the Sas2 protein and found that it also contains the protein products of the SAS4 and SAS5 genes. While acetyltransferase activity of this SAS complex has not been demonstrated, we have found that its putative HAT domain is required for its function in gene silencing in vivo. Surprisingly, the Sas4 component of this complex mediates interactions of the complex with a protein known as Asf1 (anti-silencing function 1). This protein is a histone chaperone/chromatin assembly protein that can interfere with gene silencing when overexpressed in yeast cells. We have found that deletion of ASF1 results in silencing defects similar to the loss of SAS2. We are investigating the functions of the SAS complex and Asf1 in both chromatin assembly and gene silencing.
Studies on the recruitment of SAGA, NuA4, and SWI/SNF are supported by a grant from the National Institute of General Medical Sciences.
Last updated October 12, 2001
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