Jerry L. Workman

The Role of Chromatin during Transcription

Chromatin structure imposes significant obstacles on all aspects of transcription that are mediated by RNA polymerase II. The dynamics of chromatin structure are tightly regulated through multiple mechanisms including histone modification, chromatin remodeling, histone variant incorporation, and histone eviction. In this Review, we highlight advances in our understanding of chromatin regulation and discuss how such regulation affects the binding of transcription factors as well as the initiation and elongation steps of transcription.

Histone H3 methylation by Set2 directs deacetylation of coding regions by Rpd3S to suppress spurious intragenic transcription

Yeast Rpd3 histone deacetylase plays an important role at actively transcribed genes. We characterized two distinct Rpd3 complexes, Rpd3L and Rpd3S, by MudPIT analysis. Both complexes shared a three subunit core and Rpd3L contains unique subunits consistent with being a promoter targeted corepressor. Rco1 and Eaf3 were subunits specific to Rpd3S. Mutants of RCO1 and EAF3 exhibited increased acetylation in the FLO8 and STE11 open reading frames (ORFs) and the appearance of aberrant transcripts initiating within the body of these ORFs. Mutants in the RNA polymerase II-associated SET2 histone methyltransferase also displayed these defects. Set2 functioned upstream of Rpd3S and the Eaf3 methyl-histone binding chromodomain was important for recruitment of Rpd3S and for deacetylation within the STE11 ORF. These data indicate that Pol II-associated Set2 methylates H3 providing a transcriptional memory which signals for deacetylation of ORFs by Rpd3S. This erases transcription elongation-associated acetylation to suppress intragenic transcription initiation.

Histone acetyltransferase complexes: one size doesn't fit all

Over the past 10 years, the study of histone acetyltransferases (HATs) has advanced significantly, and a number of HATs have been isolated from various organisms. It emerged that HATs are highly diverse and generally contain multiple subunits. The functions of the catalytic subunit depend largely on the context of the other subunits in the complex. We are just beginning to understand the specialized roles of HAT complexes in chromosome decondensation, DNA-damage repair and the modification of non-histone substrates, as well as their role in the broader epigenetic landscape, including the role of protein domains within HAT complexes and the dynamic interplay between HAT complexes and existing histone modifications.

The TAFII250 Subunit of TFIID Has Histone Acetyltransferase Activity

The transcription initiation factor TFIID is a multimeric protein complex composed of TATA box-binding protein (TBP) and many TBP-associated factors (TAF(II)s). TAF(II)s are important cofactors that mediate activated transcription by providing interaction sites for distinct activators. Here, we present evidence that human TAF(II)250 and its homologs in Drosophila and yeast have histone acetyltransferase (HAT) activity in vitro. HAT activity maps to the central, most conserved portion of dTAF(II)230 and yTAF(II)130. The HAT activity of dTAF(II)230 resembles that of yeast and human GCN5 in that it is specific for histones H3 and H4 in vitro. Our findings suggest that targeted histone acetylation at specific promoters by TAF(II)250 may be involved in mechanisms by which TFIID gains access to transcriptionally repressed chromatin.

Purification and biochemical heterogeneity of the mammalian SWI-SNF complex.

We have purified distinct complexes of nine to 12 proteins [referred to as BRG1‐associated factors (BAFs)] from several mammalian cell lines using an antibody to the SWI2‐SNF2 homolog BRG1. Microsequencing revealed that the 47 kDa BAF is identical to INI1. Previously INI1 has been shown to interact with and activate human immunodeficiency virus integrase and to be homologous to the yeast SNF5 gene. A group of BAF47‐associated proteins were affinity purified with antibodies against INI1/BAF47 and were found to be identical to those co‐purified with BRG1, strongly indicating that this group of proteins associates tightly and is likely to be the mammalian equivalent of the yeast SWI‐SNF complex. Complexes containing BRG1 can disrupt nucleosomes and facilitate the binding of GAL4‐VP16 to a nucleosomal template similar to the yeast SWI‐SNF complex. Purification of the complex from several cell lines demonstrates that it is heterogeneous with respect to subunit composition. The two SWI‐SNF2 homologs, BRG1 and hbrm, were found in separate complexes. Certain cell lines completely lack BRG1 and hbrm, indicating that they are not essential for cell viability and that the mammalian SWI‐SNF complex may be tailored to the needs of a differentiated cell type.

ATP-Dependent Chromatin-Remodeling Complexes

The importance of histones and chromatin structure in the regulation of eukaryotic gene transcription has become much more widely accepted over the past few years. It has been clear for a decade that histones contribute to the regulation of transcription both in vitro and in vivo (reviewed in references 14, 34, 50, 64, and 120). More recent studies have led to the striking observation that several protein complexes involved in transcription regulation can function, at least in part, by modifying histones or altering chromatin structure (for recent reviews, see references 3, 44, 49, 51, 52, 87, 100, and 119). While it is clear that many of these protein complexes have functions in addition to chromatin modification, they illustrate the importance of chromatin structure as a part of transcription regulation mechanisms. The most widely characterized chromatin-modifying complexes studied to date can be classified into two major groups, based on their modes of action, as follows: (i) ATP-dependent complexes, which use the energy of ATP hydrolysis to locally disrupt or alter the association of histones with DNA, and (ii) histone acetyltransferase (HAT) and histone deacetylase (HDAC) complexes, which regulate the transcriptional activity of genes by determining the level of acetylation of the amino-terminal domains of nucleosomal histones associated with them. This review will focus primarily on the ATP-dependent remodeling complexes. For recent reviews of HAT and HDAC complexes, see references 5, 20, 31, and 55. Here we provide an organized listing of the ATP-dependent chromatin-remodeling complexes described to date and illustrate the relationships between their subunits. We also review the data available with regard to their mechanisms of action and promoter targeting as well as regulation of their activity. Finally, we examine the relationship between these complexes and the HAT complexes.

Histone ubiquitination: triggering gene activity.

Recently, many of the enzymes responsible for the addition and removal of ubiquitin from the histones H2A and H2B have been identified and characterized. From these studies, it has become clear that H2A and H2B ubiquitination play critical roles in regulating many processes within the nucleus, including transcription initiation and elongation, silencing, and DNA repair. In this review, we present the enzymes involved in H2A and H2B ubiquitination and discuss new evidence that links histone ubiquitination to other chromatin modifications, which has provided a model for the role of H2B ubiquitination, in particular, in transcription initiation and elongation.

Transcriptional activators direct histone acetyltransferase complexes to nucleosomes

Transcriptional co-activators were originally identified as proteins that act as intermediaries between upstream activators and the basal transcription machinery. The discovery that co-activators such as Tetrahymena and yeast Gcn5,, as well as human p300/CBP,, pCAF, Src-1, ACTR and TAFII250, can acetylate histones suggests that activators may be involved in targeting acetylation activity to promoters. Several histone deacetylases have been linked to transcriptional co-repressor proteins, suggesting that the action of both acetylases and deacetylases is important in the regulation of many genes. Here we demonstrate the binding of two native yeast histone acetyltransferase (HAT) complexes to the herpesvirus VP16 activation domain and the yeast transcriptional activator Gcn4, and show that it is their interaction with the VP16 activation domain that targets Gal4–VP16-bound nucleosomes for acetylation. We find that Gal4–VP16-driven transcription from chromatin templates is stimulated by both HAT complexes in an acetyl CoA-dependent reaction. Our results demonstrate the targeting of native HAT complexes by a transcription-activation domain to nucleosomes in order to activate transcription.

The diverse functions of histone acetyltransferase complexes

Although histone acetylation has historically been linked to transcription activation, recent studies indicate that this modification and the enzymes that catalyze it have much broader and diverse functions. Histone acetyltransferase complexes are involved in such diverse processes as transcription activation, gene silencing, DNA repair and cell-cycle progression. The high conservation of the acetyltransferase complexes and their functions illustrates their central role in cell growth and development.

Promoter targeting and chromatin remodeling by the SWI/SNF complex

The SWI/SNF complex is a 2 MDa multi-subunit DNA-dependent ATPase that contributes to the regulation of gene transcription by altering chromatin structure. Recent studies have revealed that the SWI/SNF complex is targeted to promoters via direct interactions with transcription activators and have provided insights into mechanisms by which the complex alters nucleosome structure and contributes to the remodeling of chromatin.