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In this issue, Hughes et al. (2012) show that nucleosome positioning is determined not by any single mechanism, but by the coordinated action of multiple factors, including the underlying DNA sequence, species-specific DNA binding proteins, chromatin remodelers, and the transcription machinery.

The SAGA complex is a conserved histone acetyltransferase-coactivator that regulates gene expression in Saccharomyces cerevisiae. SAGA contains a number of subunits known to function in transcription including Spt and Ada proteins, the Gcn5 acetyltransferase, a subset of TATA-binding-protein-associated factors (TAF(II)s), and Tra1. Here we report the identification of SLIK (SAGA-like), a complex related in composition to SAGA. Notably SLIK uniquely contains the protein Rtg2, linking the function of SLIK to the retrograde response pathway. Yeast harboring mutations in both SAGA and SLIK complexes displays synthetic phenotypes more severe than those of yeast with mutation of either complex alone. We present data indicating that distinct forms of the SAGA complex may regulate specific subsets of genes and that SAGA and SLIK have multiple partly overlapping activities, which play a critical role in transcription by RNA polymerase II.

Several yeast transcription activators have been shown to interact with and recruit histone acetyltransferase complexes to promoters in chromatin. The promiscuity of activator/HAT interactions suggests that additional factors temporally regulate these interactions in response to signaling pathways. In this study, we demonstrate that the negative regulator, Gal80, blocks interactions between the SAGA and NuA4 HAT complexes and the Gal4 activator. By contrast, Gal80 did not inhibit SAGA and NuA4 interaction with another activator Gcn4. The function of Gal80 prevented Gal4 targeting of SAGA and displaced SAGA targeted by Gal4 to a promoter within a nucleosome array. In the same set of experiments, targeting of SAGA by Gcn4 was unaffected by Gal80. These studies demonstrate that the specificity of HAT/activator interactions can be dictated by cofactors that modulate activation domain function in response to cellular signals.

Histone H3 lysine 4 trimethylation needed for transcription is mediated by the Set1 methyltransferase and requires prior monoubiquitination of histone H2B. In this issue, Latham et al. (2011) report that dimethylation of the yeast kinetochore protein Dam1 by Set1 similarly requires H2B monoubiquitination. Thus, H2B ubiquitination signals for methylation beyond chromatin.

Recent discoveries of histone demethylases have shown that the dynamic regulation of histone methylation is important in differentiation and development. A paper in this issue of The EMBO Journal demonstrates that an H3K4me3 demethylase, KDM5B, is required for the regulation of self‐renewal and pluripotency of embryonic stem (ES) cells by removing intragenic H3K4me3 and repressing cryptic transcription.

Spinocerebellar ataxia 7 (SCA7) is an incurable disease caused by expansion of CAG trinucleotide sequences within the Ataxin-7 gene. This elongated CAG tract results in an Ataxin-7 protein bearing an expanded polyglutamine (PolyQ) repeat. SCA7 disease is characterized by progressive neural and retinal degeneration leading to ataxia and blindness. Evidence gathered from investigating SCA7 and other PolyQ diseases strongly suggest that misregulation of gene expression contributes to neurodegeneration. In fact, Ataxin-7 is a subunit of the essential Spt-Ada-Gcn5-Acetltransferase (SAGA) chromatin modifying complex that regulates expression of a large number of genes. Here we discuss recent insights into Ataxin-7 function and, considering these findings, propose a model for how polyglutamine expansion of Ataxin-7 may affect Ataxin-7 function to alter chromatin modifications and gene expression.

It is well established that acetylation of histone and nonhistone proteins is intimately linked to transcriptional activation. However, loss of acetyltransferase activity has also been shown to cause silencing defects, implicating acetylation in gene silencing. The something about silencing (Sas) 2 protein of Saccharomyces cerevisiae, a member of the MYST (MOZ,Ybf2/Sas3, Sas2, and TIP60) acetyltransferase family, promotes silencing at HML and telomeres. Here we identify a ∼450-kD SAS complex containing Sas2p, Sas4p, and the tf2f-related Sas5 protein. Mutations in the conserved acetyl-CoA binding motif of Sas2p are shown to disrupt the ability of Sas2p to mediate the silencing at HML and telomeres, providing evidence for an important role for the acetyltransferase activity of the SAS complex in silencing. Furthermore, the SAS complex is found to interact with chromatin assembly factor Asf1p, and asf1 mutants show silencing defects similar to mutants in the SAS complex. Thus, ASF1-dependent chromatin assembly may mediate the role of the SAS complex in silencing.

Promoter-specific recruitment of histone acetyltransferase activity is often critical for transcriptional activation. We present a detailed study of the interaction between the histone acetyltransferase complexes SAGA and NuA4, and transcription activators. We demonstrate by affinity chromatography and photo–cross-linking label transfer that acidic activators directly interact with Tra1p, a shared subunit of SAGA and NuA4. Mutations within the COOH-terminus of Tra1p disrupted its interaction with activators and resulted in gene-specific transcriptional defects that correlated with lowered promoter-specific histone acetylation. These data demonstrate that the essential Tra1 protein serves as a common target for activators in both SAGA and NuA4 acetyltransferases.

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 mechansims by which the complex alters nucleosome structure and contributes to the remodeling of chromatin.

The SWI-SNF complex has been shown to alter nucleosome conformation in an ATP-dependent manner, leading to increased accessibility of nucleosomal DNA to transcription factors. In this study, we show that the SWI-SNF complex can potentiate the activity of the glucocorticoid receptor (GR) through the N-terminal transactivation domain, tau1, in both yeast and mammalian cells. GR-tau1 can directly interact with purified SWI-SNF complex, and mutations in tau1 that affect the transactivation activity in vivo also directly affect tau1 interaction with SWI-SNF. Furthermore, the SWI-SNF complex can stimulate tau1-driven transcription from chromatin templates in vitro. Taken together, these results support a model in which the GR can directly recruit the SWI-SNF complex to target promoters during glucocorticoid-dependent gene activation. We also provide evidence that the SWI-SNF and SAGA complexes represent independent pathways of tau1-mediated activation but play overlapping roles that are able to compensate for one another under some conditions.

Histone acetylation is closely linked to gene transcription. The identification of histone acetyltransferases (HATs) and the large multiprotein complexes in which they reside has yielded important insights into how these enzymes regulate transcription. The demonstration that HAT complexes interact with sequence-specific activator proteins illustrates how these complexes target specific genes. In addition to histones, some HATs can acetylate non-histone proteins suggesting multiple roles for these enzymes.

SDS3 (suppressor of defective silencing 3) was originally identified in a screen for mutations that cause increased silencing of a crippled HMR silencer in a rap1 mutant background. In addition, sds3 mutants have phenotypes very similar to those seen in sin3 and rpd3 mutants, suggesting that it functions in the same genetic pathway. In this manuscript we demonstrate that Sds3p is an integral subunit of a previously identified high molecular weight Rpd3p.Sin3p containing yeast histone deacetylase complex. By analyzing an sds3Delta strain we show that, in the absence of Sds3p, Sin3p can be chromatographically separated from Rpd3p, indicating that Sds3p promotes the integrity of the complex. Moreover, the remaining Rpd3p complex in the sds3Delta strain had little or no histone deacetylase activity. Thus, Sds3p plays important roles in the integrity and catalytic activity of the Rpd3p.Sin3p complex.

Post-translational acetylation of histone H4 N-terminal tail in chromatin has been associated with several nuclear processes including transcription. We report the purification and characterization of a native multisubunit complex (NuA4) from yeast that acetylates nucleosomal histone H4. NuA4 has an apparent molecular mass of 1.3 MDa. All four conserved lysines of histone H4 can be acetylated by NuA4. We have identified the catalytic subunit of the complex as the product of ESA1, an essential gene required for cell cycle progression in yeast. Antibodies against Esa1p specifically immunoprecipitate NuA4 activity whereas the complex purified from a temperature-sensitive esa1 mutant loses its acetyltransferase activity at the restrictive temperature. Additionally, we have identified another subunit of the complex as the product of TRA1, an ATM-related essential gene homologous to human TRRAP, an essential cofactor for c-Myc- and E2F-mediated oncogenic transformation. Finally, the ability of NuA4 to stimulate GAL4-VP16-driven transcription from chromatin templates in vitro is also lost in the temperature-sensitive esa1 mutant. The function of the essential Esa1 protein as the HAT subunit of NuA4 and the presence of Tra1p, a putative transcription activator-interacting subunit, supports an essential link between nuclear H4 acetylation, transcriptional regulation and cell cycle control.

The class I helix-loop-helix (HLH) proteins, which include E2A, HEB, and E2-2, have been shown to be required for lineage-specific gene expression during T and B lymphocyte development. Additionally, the E2A proteins function to regulate V(D)J recombination, possibly by allowing access of variable region segments to the recombination machinery. The mechanisms by which E2A regulates transcription and recombination, however, are largely unknown. Here, we identify a novel motif, LDFS, present in the vertebrate class I HLH proteins as well as in a yeast HLH protein that is essential for transactivation. We provide both genetic and biochemical evidence that the highly conserved LDFS motif stimulates transcription by direct recruitment of the SAGA histone acetyltransferase complex.

Recently, we reported the identification of a 55-kDa polypeptide (p55) from Tetrahymena macronuclei as a catalytic subunit of a transcription-associated histone acetyltransferase (HAT A). Extensive homology between p55 and Gcn5p, a component of the SAGA and ADA transcriptional coactivator complexes in budding yeast, suggests an immediate link between the regulation of chromatin structure and transcriptional output. Here we report the characterization of a second transcription-associated HAT activity from Tetrahymena macronuclei. This novel activity is distinct from complexes containing p55 and putative ciliate SAGA and ADA components and shares several characteristics with NuA4 (for nucleosomal H2A/H4), a 1.8-MDa, Gcn5p-independent HAT complex recently described in yeast. A key feature of both the NuA4 and Tetrahymena activities is their acetylation site specificity for lysines 5, 8, 12, and 16 of H4 and lysines 5 and 9 of H2A in nucleosomal substrates, patterns that are distinct from those of known Gcn5p family members. Moreover, like NuA4, the Tetrahymena activity is capable of activating transcription from nucleosomal templates in vitro in an acetyl coenzyme A-dependent fashion. Unlike NuA4, however, sucrose gradient analyses of the ciliate enzyme, following sequential denaturation and renaturation, estimate the molecular size of the catalytically active subunit to be approximately 80 kDa, consistent with the notion that a single polypeptide or a stable subcomplex is sufficient for this H2A/H4 nucleosomal HAT activity. Together, these data document the importance of this novel HAT activity for transcriptional activation from chromatin templates and suggest that a second catalytic HAT subunit, in addition to p55/Gcn5p, is conserved between yeast and Tetrahymena.

The coactivator/adaptor protein Gcn5 is a conserved histone acetyltransferase, which functions as the catalytic subunit in multiple yeast transcriptional regulatory complexes. The ability of Gcn5 to acetylate nucleosomal histones is significantly reduced relative to its activity on free histones, where it predominantly modifies histone H3 at lysine 14. However, the association of Gcn5 in multisubunit complexes potentiates its nucleosomal histone acetyltransferase activity. Here, we show that the association of Gcn5 with other proteins in two native yeast complexes, Ada and SAGA (Spt-Ada-Gcn5-acetyltransferase), directly confers upon Gcn5 the ability to acetylate an expanded set of lysines on H3. Furthermore Ada and SAGA have overlapping, yet distinct, patterns of acetylation, suggesting that the association of specific subunits determines site specificity.

To understand the mechanisms by which the chromatin-remodeling SWI/SNF complex interacts with DNA and alters nucleosome organization, we have imaged the SWI/SNF complex with both naked DNA and nucleosomal arrays by using energy-filtered microscopy. By making ATP-independent contacts with DNA at multiple sites on its surface, SWI/SNF creates loops, bringing otherwise-distant sites into close proximity. In the presence of ATP, SWI/SNF action leads to the disruption of nucleosomes within domains that appear to be topologically constrained by the complex. The data indicate that the action of one SWI/SNF complex on an array of nucleosomes can lead to the formation of a region where multiple nucleosomes are disrupted. Importantly, nucleosome disruption by SWI/SNF results in a loss of DNA content from the nucleosomes. This indicates a mechanism by which SWI/SNF unwraps part of the nucleosomal DNA.

Recent progress in identifying the catalytic subunits of histone acetyltransferase (HAT) complexes has implicated histone acetylation in the regulation of transcription. Here, we have analyzed the function of two native yeast HAT complexes, SAGA (Spt-Ada-Gcn5 Acetyltransferase) and NuA4 (nucleosome acetyltransferase of H4), in activating transcription from preassembled nucleosomal array templates in vitro. Each complex was tested for the ability to enhance transcription driven by GAL4 derivatives containing either acidic, glutamine-rich, or proline-rich activation domains. On nucleosomal array templates, the SAGA complex selectively stimulates transcription driven by the VP16 acidic activation domain in an acetyl coenzyme A-dependent manner. In contrast, the NuA4 complex facilitates transcription mediated by any of the activation domains tested if allowed to preacetylate the nucleosomal template, indicating a general stimulatory effect of histone H4 acetylation. However, when the extent of acetylation by NuA4 is limited, the complex also preferentially stimulates VP16-driven transcription. SAGA and NuA4 interact directly with the VP16 activation domain but not with a glutamine-rich or proline-rich activation domain. These data suggest that recruitment of the SAGA and NuA4 HAT complexes by the VP16 activation domain contributes to HAT-dependent activation. In addition, extensive H4/H2B acetylation by NuA4 leads to a general activation of transcription, which is independent of activator-NuA4 interactions.

Many studies have linked acetylation of lysine residues on the amino-terminal tails of the core histones to transcriptional activity of cellular chromatin. New insights into this field were gained on the identification of the first nuclear, type A histone acetyltransferase (HAT). The yeast transcriptional adaptor protein Gcn5 was identified as a nuclear HAT and thus provided a direct link between pathways of transcriptional activation and histone acetylation. However, while recombinant Gcn5 can efficiently acetylate free histone H3 and, to a lesser extent, H4 it is unable to acetylate nucleosomal histones. It is therefore very likely that additional proteins are required for Gcn5-mediated acetylation of chromosomal histones. We have recently shown that Gcn5 is the catalytic subunit of two high-molecular-weight histone acetyltransferase complexes in yeast. In addition to the Gcn5-containing ADA and SAGA HAT complexes we have identified two other HAT complexes in yeast. These are called NuA3 and NuA4 for their predominant specificity to acetylate histones H3 and H4, respectively. Here we describe the identification and characterization of four native nuclear high-molecular-weight HAT complexes in Saccharomyces cerevisiae. These purified HATs can be used in a variety of functional assays to further address questions of how acetylation has an impact on transcriptional regulation.

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.

Several previously characterized transcriptional adaptors and coactivators are now known to be histone acetyltransferases (HATs). Recent studies in Saccharomyces cerevisiae indicate that the Gcn5p HAT exists in large complexes containing several phenotypic classes of transcription factors. Genetic and biochemical studies of these transcription factors and their functions within HAT complexes suggest that acetylation of histones is one function of an integrated system of modular activities. These activities include interaction with activators, histone acetylation and interaction with basal factors. Coordination of these functions may well be an important component of gene activation in vivo.

GCN5, a putative transcriptional adapter in humans and yeast, possesses histone acetyltransferase (HAT) activity which has been linked to GCN5’s role in transcriptional activation in yeast. In this report, we demonstrate a functional interaction between human GCN5 (hGCN5) and the DNA-dependent protein kinase (DNA-PK) holoenzyme. Yeast two-hybrid screening detected an interaction between the bromodomain of hGCN5 and the p70 subunit of the human Ku heterodimer (p70-p80), which is the DNA-binding component of DNA-PK. Interaction between intact hGCN5 and Ku70 was shown biochemically using recombinant proteins and by coimmunoprecipitation of endogenous proteins following chromatography of HeLa nuclear extracts. We demonstrate that the catalytic subunit of DNA-PK phosphorylates hGCN5 both in vivo and in vitro and, moreover, that the phosphorylation inhibits the HAT activity of hGCN5. These findings suggest a possible regulatory mechanism of HAT activity.

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).

An early step in a pathway leading to transcriptional initiation involves the rearrangement of chromatin at gene regulatory sequences. To study this process, we have developed a biochemical system analyzing the interactions between chromatin templates composed of arrays of positioned nucleosomes and sequence-specific transcriptional activators. Here, a procedure is presented for the assembly of nucleosomal arrays on DNA fragments containing synthetic and natural gene sequences inserted within tandem repeats of sea urchin 5S rDNA. We also provide methods for the use of these templates in transcription factor-binding assays, as well as experimental data illustrating the efficacy of such analyses to uncover mechanisms directing factor-mediated nucleosome remodeling.

To investigate the potential mechanisms by which the SWI/SNF complex differentially regulates different genes we have tested whether transcription factors with diverse DNA binding domains were able to exploit nucleosome disruption by SWI/SNF. In addition to GAL4-VP16, the SWI/SNF complex stimulated nucleosome binding by the Zn2+ fingers of Sp1, the basic helix-loop-helix domain of USF, and the rel domain of NF-kappaB. In each case SWI/SNF action resulted in the formation of a stable factor-nucleosome complex that persisted after detachment of SWI/SNF from the nucleosome. Thus, stimulation of factor binding by SWI/SNF appears to be universal. The degree of SWI/SNF stimulation of nucleosome binding by a factor appears to be inversely related to the extent that binding is inhibited by the histone octamer. Cooperative binding of 5 GAL4-VP16 dimers to a 5-site nucleosome enhanced GAL4 binding relative to a single-site nucleosome, but this also reduced the degree of stimulation by SWI/SNF. The SWI/SNF complex increased the affinity of 5 GAL4-VP16 dimers for nucleosomes equal to that of DNA but no further. Similarly, multimerized NF-kappaB sites enhanced nucleosome binding by NF-kappaB and reduced the stimulatory effect of SWI/SNF. Thus, cooperative binding of factors to nucleosomes is partially redundant with the function of the SWI/SNF complex.

During the process of transcription elongation, the chromatin structure of transcribed sequences can be perturbed, exposing cryptic promoter-like sequences within the body of transcribed genes to function as initiation sites. Re-establishing a stable repressive structure of open reading frames requires histone chaperones, methyltransferases, deacetylases and chromatin remodeling complexes. The Set2/Rpd3S pathway is used by elongating RNA polymerase II to signal for histone deacetylation in its wake. Set2 associates with the elongating form of RNA polymerase II and co-transcriptionally methylates histone H3K36. H3K36 is recognized by the Rpd3S deacetylase complex to deacetylate histones in transcribed sequences. In recent work, we have found that a major source of cotranscriptional histone acetylation is the incorporation of soluble, Rtt109 H3K56-acetylated histones by the Asf1 histone chaperone. Set2 methylation of H3K36 promotes retention of the original histones and suppresses the incorporation of soluble histones by Asf1. By identifying factors that interact with H3K36 methylated nucleosomes, we have found that chromatin remodeling is also required to stabilize the chromatin structures of open reading frames following transcription elongation. Our studies identified the ATP-dependent chromatin remodelers Isw1 and Chd1 as two factors involved in this pathway as deletion of ISW1 and CHD1 enhanced the cryptic transcript phenotype caused by set2. Moreover, loss of ISW1 and CHD1 also enhanced the incorporation of new histones from the soluble pool into chromatin. Thus, retention of original histones, deacetylation of any new ones and their organization by chromatin remodeling are all required to re-establish stable chromatin resistant to cryptic transcription initiation.

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.

To investigate the mechanisms by which transcription factors invade nucleosomal DNA and replace histones at control elements, we have examined the response of the histone octamer to transcription factor binding in the presence of histone-binding proteins (i.e., nucleosome assembly factors). We found that yeast nucleosome assembly protein 1 (NAP-1) stimulated transcription factor binding and nucleosome displacement in a manner similar to that of nucleoplasmin. In addition, disruption of the histone octamer was required both for the stimulation of transcription factor binding to nucleosomal DNA and for transcription factor-induced nucleosome displacement mediated by nucleoplasmin or NAP-1. While NAP-1 and nucleoplasmin stimulated the binding of a fusion protein (GAL4-AH) to control nucleosome cores, this stimulation was lost upon covalent histone-histone cross-linking within the histone octamers. In addition, both NAP-1 and nucleoplasmin were able to mediate histone displacement upon the binding of five GAL4-AH dimers to control nucleosome cores; however, this activity was also forfeited when the histone octamers were cross-linked. These data indicate that octamer disruption is required for both stimulation of factor binding and factor-dependent histone displacement by nucleoplasmin and NAP-1. By contrast, transcription factor-induced histone transfer onto nonspecific competitor DNA did not require disruption of the histone octamer. Thus, histone displacement in this instance occurred by transfer of complete histone octamers, a mechanism distinct from that mediated by the histone-binding proteins nucleoplasmin and NAP-1.

To investigate mechanisms by which multiple transcription factors access complex promoters and enhancers within cellular chromatin, we have analyzed the binding of disparate factors to nucleosome cores. We used a purified in vitro system to analyze binding of four activator proteins, two GAL4 derivatives, USF, and NF-kappa B (KBF1), to reconstituted nucleosome cores containing different combinations of binding sites. Here we show that binding of any two or all three of these factors to nucleosomal DNA is inherently cooperative. Thus, the binuclear Zn clusters of GAL4, the helix-loop-helix/basic domains of USF, and the rel domain of NF-kappa B all participated in cooperative nucleosome binding, illustrating that this effect is not restricted to a particular DNA-binding domain. Simultaneous binding by two factors increased the affinity of individual factors for nucleosomal DNA by up to 2 orders of magnitude. Importantly, cooperative binding resulted in efficient nucleosome binding by factors (USF and NF-kappa B) which independently possess little nucleosome-binding ability. The participation of GAL4 derivatives in cooperative nucleosome binding required only DNA-binding and dimerization domains, indicating that disruption of histone-DNA contacts by factor binding was responsible for the increased affinity of additional factors. Cooperative nucleosome binding required sequence-specific binding of all transcription factors, appeared to have spatial constraints, and was independent of the orientation of the binding sites on the nucleosome. These results indicate that cooperative nucleosome binding is a general mechanism that may play a significant role in loading complex enhancer and promoter elements with multiple diverse factors in chromatin and contribute to the generation of threshold responses and transcriptional synergy by multiple activator sites in vivo.

Publisher Summary This chapter discusses the basic preparations of reagents and their application to the analysis of transcription factor binding to nucleosomes. It provides protocols for the preparation of nucleosomes and histones, nucleosome reconstitution, and the analysis of transcription factor binding. The chapter also provides protocols that are applicable to any nucleosome-length DNA fragment that can be easily performed by any biochemist or molecular biologist with minimal protein experience. The only limitation to performing these experiments is the availability of sufficient quantities of the transcription factors of interest. The direct analysis of transcription factor binding to nucleosomes has revealed many general properties of factor-nucleosome interactions. Different factors are inhibited in binding to different degrees by the occupancy of their binding sites in nucleosomes. For example, the glucocorticoid receptor exhibits very efficient nucleosome binding abilities, whereas similar analyses indicate that the binding of NF1 and the human heat-shock factor are severely inhibited by nucleosomes. Other factors demonstrate intermediate levels of affinity for nucleosomal DNA.

Publisher Summary This chapter describes advanced techniques to analyze the interactions of an individual or group of factors with nucleosome cores. The methods presented in the chapter allow an investigation of the parameters governing the binding of a particular factor to nucleosomes, including nucleosome position effects and the role of the core histone amino termini. In addition, approaches to analyze the cooperativity of factor binding and the function of accessory protein complexes in factor binding are described in the chapter. It also provides protocols to directly assess the proteins present (factors and histones) in ternary complexes resulting from the binding of transcription factors to nucleosomes. These approaches can be applied to analyze the binding of an individual transcription factor. Alternatively, the methods can be used to address the function of multiple factors in binding and displacing nucleosomes reconstituted on complex enhancer or promoter elements. The histone-binding protein, nucleoplasmin, stimulates transcription factor binding to nucleosomes and can remove H2A/H2B dimers from factor/nucleosome ternary complexes.

Publisher Summary Biochemical analysis of the proteins involved in transcription by RNA polymerase II has revealed two classes of transcription factors. These include the general initiation factors, which are required in addition to RNA polymerase II for accurate initiation, and regulatory factors, which bind upstream promoter and/or enhancer elements and activate transcription initiation. Biochemical studies of the interactions of regulatory transcription factors with nucleosome cores have illustrated several parameters that restrict the binding of individual transcription factors to nucleosomal DNA. These include a differential intrinsic affinity of different factors for their recognition sites on nucleosomes, the location of the binding sites within the nucleosome core, and inhibition from the core histone amino termini. However, nucleosome-mediated repression of factor binding can be overcome by the facilitated binding of multiple factors, relief of inhibition from the core histone amino termini (that is, by histone acetylation), and through the function of accessory proteins that stimulate factor binding. The binding of regulatory factors to nucleosomes results in the formation of factor/nucleosome ternary complexes that contain bound factors, core histones, and DNA.

Recent studies suggest that the Spt16 protein of FACT shuttles H2A–H2B dimers off and on nucleosomes during transcription elongation. By restoring nucleosomes after passage of RNA polymerase II, Spt16 and Spt6 prevent transcription from cryptic promoters in coding regions that would otherwise be expressed in the absence of histones.

Histone deacetylases (HDACs) remove acetyl groups from histones and other proteins, leading to alterations in protein activity and gene expression. Drugs that target these enzymes (HDAC inhibitors or HDACis) are currently approved for the treatment of specific cancers and are under study in numerous additional clinical trials. These drugs work by causing cell cycle arrest, differentiation or apoptosis. However, less is known about their potential effects on cancer cell migration and invasion, processes implicated in metastasis. There are eleven human zinc dependent histone deacetylases and many HDACis target several of these structurally related proteins in cells. Our studies are focused on HDACs1 and 2 which reside in large protein complexes, one of which is the Sin3 complex. This complex is important for promoter-mediated histone deacetylation, transcriptional repression and cell cycle control. We previously found that specific HDACis disrupt the function of the Sin3 complex in distinct ways. Here we describe the function of a novel subunit of the Sin3 complex, a previously uncharacterized protein, named “Family with sequence similarity 60, member A” (FAM60A). We find that FAM60A represses expression of genes linked to cancer cell migration and invasion. Moreover, we show that loss of FAM60A, or a core subunit of the Sin3 complex can increase the migration of lung cancer cells. This suggests that not all HDAC-related functions are cancer promoting, and adds to data suggesting that some HDAC-related functions may act in steps in metastasis suppression pathways. This suggests that some HDACis could increase the potential for metastasis. Current studies are focused on delineating the cancer-promoting functions of the complex from those that are tumor or metastasis suppressive and testing the effects of specific HDACis on cancer cell migration and invasion. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-267. doi:1538-7445.AM2012-LB-267

In order to investigate the interrelated roles of nucleosome cores and histone H1 in transcription repression, we have employed a purified system to analyze the function of H1 in the repression of transcription factor binding to nucleosomes. H1 binding to nucleosome cores resulted in the repression of USF binding to nucleosomes. By contrast, H1 only slightly inhibited the binding of GAL4-AH, indicating that H1 differentially represses the binding of factors with different DNA-binding domains. H1-mediated repression of factor binding was dependent on the core histone amino-terminal tails. Removal of these domains alleviated H1-mediated repression and increased acetylation of these domains partly alleviated repression by H1. H1 binding assays suggest a less stable interaction of histone H1 with the core particle in the absence of the amino termini.

The binding of a GAL4-AH, USF or Sp1 to nucleosome cores was stimulated by the presence of the histone-binding protein, nucleoplasmin. Stimulation of factor binding by nucleoplasmin was specific for nucleosome reconstituted DNA and was not mimicked by non-specific histone sinks (i.e. polyglutamate or RNA). Upon GAL4-AH binding, nucleoplasmin specifically removed histones H2A and H2B from the nucleosome which enhanced the subsequent loss of the H3/H4 tetramers onto competing DNA. Thus, nucleoplasmin participated in the complete conversion of nucleosome cores to transcription factor-DNA complexes. These data indicate that proteins which bind histones can increase transcription factor binding to nucleosomal DNA and that transcription factor binding can initiate nucleosome disassembly. Similar activities of histone-binding proteins may participate in the displacement of nucleosomes at enhancers and promoters in vivo.

In order to function, transcription factors must compete for DNA binding with structural components of chromatin, including nucleosomes. Mechanisms that could be used in this competition have been characterized with the use of the DNA binding domain of the yeast GAL4 protein. The binding of GAL4 to a nucleosome core resulted in a ternary complex containing GAL4, the core histone proteins, and DNA. This ternary complex was unstable; upon the addition of nonspecific competitor DNA, it dissociated into either the original nucleosome core particle or GAL4 bound to naked DNA. Nucleosome core destabilization by GAL4 did not require a transcriptional activation domain. These data demonstrate the displacement of nucleosome cores as a direct result of binding by a regulatory factor. Similar mechanisms might affect the establishment of factor occupancy of promoters and enhancers in vivo.

Publisher Summary This chapter discusses the control of class II gene transcription during in vitro nucleosome assembly and whether the competitive nucleosome assembly pathway creates conditions under which the regulatory activity of particular factors is more apparent. In vitro functional and structural studies of nucleosome and chromatin reconstitution should allow an investigation into the individual roles of multiple factors regulating a single transcription unit. Functional studies can address the role of a factor in establishing the transcriptional potential of a promoter in chromatin and in the initiation and elongation of transcription. Binding studies can be used to address the ability of a factor to bind to nucleosomal DNA and the fate of the nucleosome upon such binding. These in vitro approaches provide assays for the function of chromosomal structural proteins in transcription. Several laboratories have used in vitro approaches to investigate the involvement of nucleosomes in class II gene transcription..

During in vitro chromatin assembly the formation of transcription complexes is in direct competition with the assembly of promoter sequences into nucleosomes. Under these conditions the fold stimulation of transcription by an upstream transcription factor (USF) was greater than that observed in the absence of nucleosome assembly. Function of USF during nucleosome assembly required the simultaneous presence of the TATA box binding protein TFIID. Unlike TFIID, USF alone was unable to prevent repression of the promoter during nucleosome assembly. Furthermore, USF displayed reduced or no transcriptional stimulatory activity when added to previously assembled minichromosomes. Under conditions of nucleosome assembly, USF increased the number of assembled minichromosomes which contained stable preinitiation complexes. Subsequent to assembly, the rate at which preformed complexes initiated transcription appeared to be independent of the presence of USF. Thus USF potentiated the subsequent transcriptional activity of the promoter indirectly, apparently by increasing the rate or stability of TFIID binding. This activity resulted in the promoter becoming resistant to nucleosome mediated repression. These observations suggest that some ubiquitous upstream factors, e.g. USF, may play an important role in establishing the transcriptional potential of cellular genes during chromatin assembly.

DNA is not stuffed randomly into the nucleus of cells. Rather, it is neatly coiled around a series of histone octamers. John and Workman discuss how the cell transcribes RNA messages from such a seemingly inaccessible template. They describe a report in this issue by [ LeRoy et al ][1]. that identifies several proteins that help to move the nucleosomes out of the way of the transcription machinery. [1]: http://www.sciencemag.org/cgi/content/short/282/5395/1900

Does it or doesn't it? For some time there has been controversy as to whether TBP (TATA-binding protein) needs TAFs (TBP-associated factors) to do its crucial job in gene transcription. Five papers now add to this debate — they show that TAFs have a broader function in gene expression than was previously thought.

Acetylation at histone H4 lysine 16 is involved in many cellular processes in organisms as diverse as yeast and humans. A recent biochemical study pinpoints this particular acetylation mark as a switch for changing chromatin from a repressive to a transcriptionally active state.

A report on the FASEB Summer Research Conference 'Chromatin and Transcription', Snowmass, USA, 9-14 July 2005.

A report on the American Society for Biochemistry and Molecular Biology symposium 'Transcriptional Regulation by Chromatin and RNA polymerase II', Lake Tahoe, USA, 29 October-1 November 2004.

Recent studies show that active regulatory regions of the yeast genome have a lower density of nucleosomes than other regions, and that there is an inverse correlation between nucleosome density and the transcription rate of a gene. This may be the result of transcription factors displacing nucleosomes.