Distribution of acetylated histones resulting from Gal4-VP16 recruitment of SAGA and NuA4 complexes.

We analyzed the targeting of histone acetyltransferase (HAT) complexes by DNA-binding activators during transcriptional activation and the resulting distribution of acetylated histones. An in vitro competition assay was developed to acetylate and transcribe a nucleosomal array template in the presence of excess non-specific chromatin, which mimics in vivo conditions. Stimulation of transcription from the nucleosomal array template under competitive conditions by the SAGA and NuA4 HAT complexes depended on the presence of the Gal4-VP16 activator, which recognizes sites in the promoter and directly interacts with these HATs. Importantly, the stimulation of transcription by SAGA and NuA4 depended on the presence of Gal4-VP16 during histone acetylation, and Gal4-VP16-bound nucleosomal templates were acetylated preferentially by SAGA and NuA4 relative to the competitor chromatin. While targeting of the SAGA complex led to H3 acetylation of promoter-proximal nucleosomes, targeting of the NuA4 complex led to a broader domain of H4 acetylation of >3 kbp. Thus, either promoter-proximal H3 acetylation by SAGA or broadly distributed acetylation of H4 by NuA4 activated transcription from chromatin templates.

The chromo domain protein chd1p from budding yeast is an ATP-dependent chromatin-modifying factor.

CHD proteins are members of the chromo domain family, a class of proteins involved in transcription, DNA degradation and chromatin structure. In higher eukaryotes, there are two distinct subfamilies of CHD proteins: CHD1 and CHD3/4. Analyses carried out in vitro indicate that the CHD3/4 proteins may regulate transcription via alteration of chromatin structure. However, little is known about the role of CHD proteins in vivo, particularly the CHD1 subfamily. To understand better the cellular function of CHD proteins, we initiated a study on the Chd1p protein from budding yeast. Using genomic DNA arrays, we identified genes whose expression is affected by the absence of Chd1p. A synthetic-lethal screen uncovered genetic interactions between SWI/SNF genes and CHD1. Biochemical experiments using Chd1p purified from yeast showed that it reconfigures the structure of nucleosome core particles in a manner distinct from the SWI-SNF complex. Taken together, these results suggest that Chd1p functions as a nucleosome remodeling factor, and that Chd1p may share overlapping roles with the SWI-SNF complex to regulate transcription.

AP-2 family members regulate basal and cAMP-induced expression of human chorionic gonadotropin.

The AP-2 family of transcriptional regulator proteins has three members, alpha, beta and gamma. AP-2alpha and gamma are expressed in placenta and in the human trophoblast cell line JEG-3. AP-2 has been shown to regulate expression of the placental human chorionic gonado-tropin (hCG) alpha- and beta-subunit genes, however, previous work did not distinguish between the family members. Tryptic peptides of the AP-2 protein complexes purified from JEG-3 cells by oligo-affinity chromatography using the hCGalpha AP-2 site match the amino acid sequence of AP-2gamma. The fact that AP-2gamma is present at significant levels and binds the hCGalpha trophoblast-specific element suggests that AP-2gamma is at least part of the binding complex in vivo and plays a role in regulating hCG expression. We show that mutation of each of four AP-2 binding sites within the hCGbeta promoter decreases expression in transfection assays, demonstrating that all four sites are required for maximal expression in JEG-3 cells. Furthermore, we find differences in regulation of the family members: AP-2alpha mRNA levels increase in response to cAMP while AP-2gamma mRNA levels do not. The demonstrated importance of the AP-2 sites in controlling hCGalpha and beta expression and the likely involvement of more than one family member suggest that a balance in AP-2 proteins is involved in coordinate regulation of these genes. Moreover, many placenta-restricted genes are regulated by AP-2 proteins, thus members of this family may play an important overall role in placenta-specific expression.

Transcriptional analysis of purified histone acetyltransferase complexes.

Acetylation of lysine residues within the amino-terminal tails of the core histone proteins is strongly correlated to the regulation of gene transcription in vivo. To directly study the effects of histone acetylation on transcription, we have developed a biochemical system examining the regulation of RNA polymerase II-directed transcription by native histone acetyltransferases (HATs). For the promoter sequences investigated, it has been demonstrated that HATs facilitate transcription from nucleosomal DNA templates in an acetyl-CoA-dependent fashion but do not affect transcription from histone-free templates. Here, protocols are presented describing the in vitro assembly of evenly spaced nucleosomal arrays on DNA fragments harboring gene regulatory sequences and the use of these templates with purified HAT complexes in transcription assays.

Activation domain-mediated targeting of the SWI/SNF complex to promoters stimulates transcription from nucleosome arrays.

The yeast SWI/SNF complex is required for the transcription of several yeast genes and has been shown to alter nucleosome structure in an ATP-dependent reaction. In this study, we show that the complex stimulated in vitro transcription from nucleosome templates in an activation domain-dependent manner. Transcription stimulation by SWI/SNF required an activation domain with which it directly interacts. The acidic activation domains of VP16, Gcn4, Swi5, and Hap4 interacted directly with the purified SWI/SNF complex and with the SWI/SNF complex in whole-cell extracts. The similarity of activation domain interactions and transcriptional stimulation between SWI/SNF and the SAGA histone acetyltransferase complex may account for their apparent overlapping functions in vivo.

A novel H2A/H4 nucleosomal histone acetyltransferase in Tetrahymena thermophila.

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.

Activation domain-specific and general transcription stimulation by native histone acetyltransferase complexes.

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.

Purified histone acetyltransferase complexes stimulate HIV-1 transcription from preassembled nucleosomal arrays.

Protein acetylation has been implicated in the regulation of HIV-1 gene transcription. Here, we have exploited the activities of four native histone acetyltransferase (HAT) complexes from yeast to directly test whether acetylation regulates HIV-1 transcription in vitro. HAT activities acetylating either histone H3 (SAGA, Ada, and NuA3) or H4 (NuA4) stimulate HIV-1 transcription from preassembled nucleosomal templates in an acetyl CoA-dependent manner. HIV-1 transcription from histone-free DNA is not affected by the HATs, indicating that these activities function in a chromatin-specific fashion. For Ada and NuA4, we demonstrate that acetylation of only histone proteins mediates enhanced transcription, suggesting that these complexes facilitate transcription at least in part by modifying histones. To address a potential mechanism by which HAT complexes stimulate transcription, we performed a restriction enzyme accessibility analysis. Each of the HATs increases the cutting efficiencies of restriction endonucleases targeting the HIV-1 chromatin templates in a manner not requiring transcription, suggesting that histone acetylation leads to nucleosome remodeling.

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.

A subset of TAF(II)s are integral components of the SAGA complex required for nucleosome acetylation and transcriptional stimulation.

A number of transcriptional coactivator proteins have been identified as histone acetyltransferase (HAT) proteins, providing a direct molecular basis for the coupling of histone acetylation and transcriptional activation. The yeast Spt-Ada-Gcn5-acetyltransferase (SAGA) complex requires the coactivator protein Gcn5 for HAT activity. Identification of protein subunits by mass spectrometry and immunoblotting revealed that the TATA binding protein-associated factors (TAF(II)s) TAF(II)90, -68/61, -60, -25/23, and -20/17 are integral components of this complex. In addition, TAF(II)68 was required for both SAGA-dependent nucleosomal HAT activity and transcriptional activation from chromatin templates in vitro. These results illustrate a role for certain TAF(II) proteins in the regulation of gene expression at the level of chromatin modification that is distinct from the TFIID complex and TAF(II)145.

Analysis of transcription factor-mediated remodeling of nucleosomal arrays in a purified system.

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.

Stable co-occupancy of transcription factors and histones at the HIV-1 enhancer.

To investigate mechanisms yielding DNase I-hypersensitive sites (DHSs) at gene regulatory regions, we have initiated a biochemical analysis of transcription factor binding and nucleosome remodeling with a region of the human immunodeficiency virus 1 (HIV-1) 5' long terminal repeat (LTR) that harbors constitutive DHSs in vivo. In vitro reconstitution of an HIV-1 5' LTR fragment into nucleosome core particles demonstrates that Sp1, NF-kappaB1, LEF-1, ETS-1 and USF can gain access to their binding sites in HIV-1 nucleosomal DNA. The factor-bound mononucleosomes resist histone displacement from the DNA by the chromatin remodeling activity, SW1-SNF, or the histone chaperone, nucleoplasmin, suggesting that the binding of these factors to nucleosomal HIV-1 sequences forms a stable complex that includes the underlying histones. However, when the HIV-1 5' LTR fragment is incorporated into a nucleosomal array, Sp1 and NF-kappaB1 binding produce regions of enhanced DNase I sensitivity specifically at the HIV-1 nucleosome. These regions resemble the observed in vivo DHSs, yet the HIV-1 nucleosome remains intact even in the presence of nucleoplasmin. Thus, the constitutive DHSs identified at the HIV-1 enhancer in native chromatin may reflect the presence of a ternary complex composed of transcriptional activators, histones and DNA.

Remodeling chromatin structures for transcription: what happens to the histones?

Activation of gene transcription in vivo is accompanied by an alteration of chromatin structure. The specific binding of transcriptional activators disrupts nucleosomal arrays, suggesting that the primary steps leading to transcriptional initiation involve interactions between activators and chromatin. The affinity of transcription factors for nucleosomal DNA is determined by the location of recognition sequences within nucleosomes, and by the cooperative interactions of multiple proteins targeting binding sites contained within the same nucleosomes. In addition, two distinct types of enzymatic complexes facilitate binding of transcription factors to nucleosomal DNA. These include type A histone acetyltransferases (e.g. GCN5/ADA transcriptional adaptor complex) and ATP-driven molecular machines that disrupt histone-DNA interactions (e.g. SWI/SNF and NURF complexes). These observations raise the important question of what happens to the histones during chromatin remodeling. We discuss evidence supporting the retention of histones at transcription factor-bound sequences as well as two alternative pathways of histone loss from gene control elements upon transcription factor binding: histone octamer sliding and histone dissociation.

GATA-binding proteins regulate the human gonadotropin alpha-subunit gene in the placenta and pituitary gland.

The human glycoprotein hormone alpha-subunit gene is expressed in two quite dissimilar tissues, the placenta and anterior pituitary. Tissue-specific expression is determined by combinations of elements, some of which are common and others of which are specific to each tissue. In the placenta, a composite enhancer confers specific expression. It contains four protein-binding sites: two cyclic AMP (cAMP) response elements that bind CREB, a trophoblast-specific element that binds TSEB, and a sequence motif, AGATAA, that matches the consensus binding site for a family of transcription factors termed the GATA-binding proteins. In pituitary gonadotropes, the cAMP response elements remain important for expression, TSEB is absent, and elements further upstream participate in tissue-specific expression. Here we establish a regulatory role for the GATA element in both the placenta and pituitary by demonstrating that a mutation of this element decreases alpha-subunit gene expression 15-fold in JEG-3 human placental cells and 2.5-fold in alpha T3-1 mouse pituitary gonadotropes. In JEG-3 cells, human GATA-2 (hGATA-2) and hGATA-3 are highly expressed and both proteins bind to the alpha-subunit gene GATA element. In alpha T3-1 cells, the GATA motif is bound by mouse GATA-2 (mGATA-2) and an mGATA-4-related protein. Cotransfection of hGATA-2 or hGATA-3 into alpha T3-1 cells activates the alpha-subunit gene threefold. These studies establish a role for the GATA-binding proteins in placental and pituitary alpha-subunit gene expression, significantly expanding the known target genes of GATA-2, GATA-3, and perhaps GATA-4.

Coordinate control of the alpha- and beta-subunit genes of human chorionic gonadotropin by trophoblast-specific element-binding protein.

The alpha- and beta-subunit genes of hCG are coordinately regulated in the trophectoderm of the early embryo and placenta. Placenta-specific expression of the alpha-subunit gene is determined by a composite enhancer made of three clustered components: cAMP-responsive elements, a GATA site, and the trophoblast-specific element (TSE). We have investigated the basis of placenta-specific expression of the major hCG beta-subunit gene, hCG beta 5. Enhancement of expression localizes to the region from -305 to -279, whereas full cAMP regulation requires the region from -305 to -249. Four DNAse-I footprints are present, three of which can be competed by the TSE element from the alpha-subunit gene. Methylation interference establishes that binding to the element located in the key region for expression, from -301 to -275, requires contacts with a CCNNNGGG core sequence that matches the alpha-subunit gene TSE. Sequence-specific DNA affinity chromatography using the alpha-subunit gene TSE allows purification of TSE-binding protein. This purified protein binds specifically to the key element, -301 to -275, and to at least two additional TSE elements clustered in the regulatory region of the hCG beta 5 gene. We conclude that both the alpha- and beta-subunit genes of hCG require the placenta-specific factor TSE-binding protein for expression, providing a mechanism for their coordinate regulation in placental cells.

Evolution of placenta-specific gene expression: comparison of the equine and human gonadotropin alpha-subunit genes.

Primate and equine species are thought to be unique among mammals in synthesizing placental gonadotropin glycoprotein hormones. Human chorionic gonadotropin (CG) and equine pregnant mare's serum gonadotropin (PMSG) are produced in placenta by the specific activation of a glycoprotein hormone alpha-subunit gene and a corresponding beta-subunit gene. The evolutionary mechanisms for the apparently independent acquisition of tissue specificity were investigated by cloning the 5' flanking region of the equine alpha-subunit gene and comparing the DNA elements and trans-acting factors involved in placental expression. We find that though the equine gene is expressed and induced by cAMP, it does not contain the elements known to confer tissue-specific expression to the human gene, the cAMP response element (CRE) and the trophoblast-specific element (TSE), nor does it bind to the trans-acting factors CREB and TSEB. Instead, an additional factor (alpha-ACT) is found which binds to the equine and human, but not the murine, alpha-subunit genes in a region between the positions of the CRE and TSE and confers cAMP responsiveness.