Increasing the rate of chromatin remodeling and gene activation--a novel role for the histone acetyltransferase Gcn5.

Histone acetyltransferases (HATs) such as Gcn5 play a role in transcriptional activation. However, the majority of constitutive genes show no requirement for GCN5, and even regulated genes, such as the yeast PHO5 gene, do not seem to be affected significantly by its absence under normal activation conditions. Here we show that even though the steady-state level of activated PHO5 transcription is not affected by deletion of GCN5, the rate of activation following phosphate starvation is significantly decreased. This delay in transcriptional activation is specifically due to slow chromatin remodeling of the PHO5 promoter, whereas the transmission of the phosphate starvation signal to the PHO5 promoter progresses at a normal rate. Chromatin remodeling is equally delayed in a galactose-inducible PHO5 promoter variant in which the Pho4 binding sites have been replaced by Gal4 binding sites. By contrast, activation of the GAL1 gene by galactose addition occurs with normal kinetics. Lack of the histone H4 N-termini leads to a similar delay in activation of the PHO5 promoter. These results indicate that one important contribution of HATs is to increase the rate of gene induction by accelerating chromatin remodeling, rather than to affect the final steady-state expression levels.

A transient histone hyperacetylation signal marks nucleosomes for remodeling at the PHO8 promoter in vivo.

Chromatin remodeling of the yeast PHO8 promoter requires the SAGA histone acetyltransferase complex. We report here that SAGA is necessary and sufficient to establish an activator-dependent hyperacetylation peak over the PHO8 promoter that is restricted to those nucleosomes that are remodeled upon activation. This local hyperacetylated state is observed upon activation in the absence of the SWI/SNF complex when the remodeling process is frozen subsequent to activator binding. Hyperacetylation is lost, however, if remodeling is permitted to go to completion. Thus, a transient histone hyperacetylation signal is shown to be a prerequisite for, and determinant of, the domain of nucleosome remodeling in vivo.

Histone acetylation and chromatin remodeling.

Chromatin represents a repressive barrier to the process of transcription. This molecular obstacle is a highly dynamic structure, able to compact the DNA of the entire genome into the confines of a nucleus, and yet it allows access to the genetic information held within. The acetylation of histones has emerged as a regulatory mechanism capable of modulating the properties of chromatin and thus the competence of the DNA template for transcriptional activation. The role of acetylation in chromatin remodeling is therefore of paramount importance to our understanding of gene regulation in vivo.

Transcriptional regulation of the yeast PHO8 promoter in comparison to the coregulated PHO5 promoter.

Expression of the PHO8 and PHO5 genes that encode a nonspecific alkaline and acid phosphatase, respectively, is regulated in response to the P(i) concentration in the medium by the same transcription factors. Upon induction by phosphate starvation, both promoters undergo characteristic chromatin remodeling, yet the extent of remodeling at the PHO8 promoter is significantly lower than at PHO5. Despite the coordinate regulation of the two promoters, the PHO8 promoter is almost 10 times weaker than PHO5. Here we show that of two Pho4 binding sites that had been previously mapped at the PHO8 promoter in vitro, only the high affinity one, UASp2, is functional in vivo. Activation of the PHO8 promoter is partially Pho2-dependent. However, unlike at PHO5, Pho4 can bind strongly to its binding site in the absence of Pho2 and remodel chromatin in a Pho2-independent manner. Replacement of the inactive UASp1 element by the UASp1 element from the PHO5 promoter results in more extensive chromatin remodeling and a concomitant 2-fold increase in promoter activity. In contrast, replacement of the high affinity UASp2 site with the corresponding site from PHO5 precludes chromatin remodeling completely and as a consequence promoter activation, despite efficient binding of Pho4 to this site. Deletion of the promoter region normally covered by nucleosomes -3 and -2 results in a 2-fold increase in promoter activity, further supporting a repressive role of these nucleosomes. These data show that there can be strong binding of Pho4 to a UAS element without any chromatin remodeling and promoter activation. The close correlation between promoter activity and the extent of chromatin disruption strongly suggests that the low level of PHO8 induction in comparison with PHO5 is partly due to the inability of Pho4 to achieve complete chromatin remodeling at this promoter.

Comparison of nucleosome remodeling by the yeast transcription factor Pho4 and the glucocorticoid receptor.

Chromatin reorganization of the PHO5 and murine mammary tumor virus (MMTV) promoters is triggered by binding of either Pho4 or the glucocorticoid receptor (GR), respectively. In order to compare the ability of Pho4 and GR to remodel chromatin and activate transcription, hybrid promoter constructs were created by insertion of the MMTV B nucleosome sequence into the PHO5 promoter and then transformed into a yeast strain expressing GR. Activation of either Pho4 (by phosphate depletion) or GR (by hormone addition) resulted in only slight induction of hybrid promoter activity. However, simultaneous activation of both Pho4 and GR resulted in synergistic activation to levels exceeding that of the wild type PHO5 promoter. Under these conditions, Pho4 completely disrupted the nucleosome containing its binding site. In contrast, GR had little effect on the stability of the MMTV B nucleosome. A minimal transactivation domain of the GR fused to the Pho4 DNA-binding domain is capable of efficiently disrupting the nucleosome with a Pho4-binding site, whereas the complementary hybrid protein (Pho4 activation domain, GR DNA-binding domain) does not labilize the B nucleosome. Therefore, we conclude that significant activation by Pho4 requires nucleosome disruption, whereas equivalent transcriptional activation by GR is not accompanied by overt perturbation of nucleosome structure. Our results show that the DNA-binding domains of the two factors play critical roles in determining how chromatin structure is modified during promoter activation.

Chromatin remodelling at the PHO8 promoter requires SWI-SNF and SAGA at a step subsequent to activator binding.

The SWI-SNF and SAGA complexes possess ATP-dependent nucleosome remodelling activity and histone acetyltransferase (HAT) activity, respectively. Mutations that eliminate the ATPase activity of the SWI-SNF complex, or the HAT activity of SAGA, abolish proper chromatin remodelling at the PHO8 promoter in vivo. These effects are mechanistically distinct, since the absence of SWI-SNF freezes chromatin in the repressed state, while the absence of Gcn5 permits a localized perturbation of chromatin structure immediately adjacent to the upstream transactivator binding site. However, this remodelling is not propagated to the proximal promoter, and no activation is observed under all conditions. Furthermore, Pho4 is bound to the PHO8 promoter in the absence of Snf2 or Gcn5, confirming a role for SWI-SNF and SAGA in chromatin remodelling independent of activator binding. These data provide new insights into the roles of the SWI-SNF and SAGA complexes in chromatin remodelling in vivo.

Requirements for chromatin modulation and transcription activation by the Pho4 acidic activation domain.

Perhaps the best characterized example of an activator-induced chromatin transition is found in the activation of the Saccharomyces cerevisiae acid phosphatase gene PHO5 by the basic helix-loop-helix (bHLH) transcription factor Pho4. Transcription activation of the PHO5 promoter by Pho4 is accompanied by the remodeling of four positioned nucleosomes which is dependent on the Pho4 activation domain but independent of transcription initiation. Whether the requirements for transcription activation through the TATA sequence are different from those necessary for the chromatin transition remains a major outstanding question. In an attempt to understand better the ability of Pho4 to activate transcription and to remodel chromatin, we have initiated a detailed characterization of the Pho4 activation domain. Using both deletion and point mutational analysis, we have defined residues between positions 75 and 99 as being both essential and sufficient to mediate transcription activation. Significantly, there is a marked concordance between the ability of mutations in the Pho4 activation domain to induce chromatin opening and transcription activation. Interestingly, the requirements for transcription activation within the Pho4 activation domain differ significantly if fused to a heterologous bHLH-leucine zipper DNA-binding domain. The implications for transcription activation by Pho4 are discussed.

Analyzing chromatin structure and transcription factor binding in yeast.

The study of chromatin, once thought to be a purely structural matrix serving to compact the DNA of the genome into the nucleus, is of increasing value for our understanding of how DNA functions in the cell. This article provides two basic procedures for the study of chromatin in vivo. The first is a DNase I-based method for the treatment of isolated nuclei to resolve the chromatin structure of a particular region; the second employs dimethyl sulfate footprinting of whole cells in vivo to determine the binding of factors to cis elements in the locus of interest. Specific examples illustrating the techniques described are given from our work on the regulation of the yeast PHO8 gene, but have also been successfully and reliably applied to the study of many other yeast loci. These procedures make it possible to correlate the binding of a transactivator with an altered or perturbed chromatin organization at a specific locus.

Absence of Gcn5 HAT activity defines a novel state in the opening of chromatin at the PHO5 promoter in yeast.

Histone acetyltransferase (HAT) activity has been demonstrated for several transcriptional activators, formally connecting chromatin modification with gene regulation. However, no effect on chromatin has been demonstrated. We have investigated the role of the HAT Gcn5 at the nucleosomally regulated PHO5 promoter. Under conditions of constitutive submaximal activation (i.e., in the absence of the negative regulator Pho80), deletion of Gcn5 determines a novel randomized nucleosomal organization across the promoter and leads to a dramatic reduction in activity. Furthermore, mutation of amino acids critical for Gcn5 HAT activity is sufficient to generate this structure. This intermediate state in chromatin opening gives way to the fully open structure upon maximal induction (phosphate starvation), even in the absence of Gcn5. Thus, Gcn5 is shown to affect directly the remodeling of chromatin in vivo.

Life with nucleosomes: chromatin remodelling in gene regulation.

In the past year, the role of chromatin has emerged at the forefront of transcription research. Discovery and characterisation of the chromatin modifying machinery have significantly advanced our understanding of the molecular activities that establish a transcriptionally competent substrate in vivo, and have underscored the importance of the part played by chromatin in the regulation of transcription.

Cooperative Pho2-Pho4 interactions at the PHO5 promoter are critical for binding of Pho4 to UASp1 and for efficient transactivation by Pho4 at UASp2.

The activation of the PHO5 gene in Saccharomyces cerevisiae in response to phosphate starvation critically depends on two transcriptional activators, the basic helix-loop-helix protein Pho4 and the homeodomain protein Pho2. Pho4 acts through two essential binding sites corresponding to the regulatory elements UASp1 and UASp2. Mutation of either of them results in a 10-fold decrease in promoter activity, and mutation of both sites renders the promoter totally uninducible. The role of Pho4 appears relatively straightforward, but the mechanism of action of Pho2 had remained elusive. By in vitro footprinting, we have recently mapped multiple Pho2 binding sites adjacent to the Pho4 sites, and by mutating them individually or in combination, we now show that each of them contributes to PHO5 promoter activity. Their function is not only to recruit Pho2 to the promoter but to allow cooperative binding of Pho4 together with Pho2. Cooperativity requires DNA binding of Pho2 to its target sites and Pho2-Pho4 interactions. A Pho4 derivative lacking the Pho2 interaction domain is unable to activate the promoter, but testing of UASp1 and UASp2 individually in a minimal CYC1 promoter reveals a striking difference between the two UAS elements. UASp1 is fully inactive, presumably because the Pho4 derivative is not recruited to its binding site. In contrast, UASp2 activates strongly in a Pho2-independent manner. From in vivo footprinting experiments and activity measurements with a promoter variant containing two UASp2 elements, we conclude that at UASp2, Pho2 is mainly required for the ability of Pho4 to transactivate.

Chromatin and transcription--how transcription factors battle with a repressive chromatin environment.

The last year has seen much progress in our understanding of chromatin and transcription. Transcriptionally active chromatin has long been correlated with a higher level of histone acetlyation. The discovery of a nuclear histone acetyltransferase activity encoded by factors with a role in transcription raises the possibility that the cell is able to dynamically modulate the (local) level of histone acteylation, switching chromatin templates from inactive to transcriptionally active states. Furthermore, histone acetylation states have shown to play a role in determining the efficacy of transcriptionally silenced chromatin in both yeast and Drosophila. The advances in our knowledge regarding the role of the origin-recognition complex in the establishment of silencing, and the requirement for a locally concentrated zone of the silence information regulator proteins in the nucleus has provided insights into the complex architecture of silenced chromatin. The goal of understanding the mechanisms by which the cell is able to 'open' repressive chromatin structures has prompted the discovery of multiple chromatin remodelling activities. These large protein complexes identified from organisms as diverse as yeast, mouse, fly and man demonstrate the ubiquity and fundamental importance of the ability to perturb the structure of chromatin allowing transcription of the desired genes. These data provide the latest and potentially most significant demonstration of the importance of the nucleosome in the regulation of transcription.

RNA polymerase II holoenzyme recruitment is sufficient to remodel chromatin at the yeast PHO5 promoter.

We examine transcriptional activation and chromatin remodeling at the PHO5 promoter in yeast by fusion proteins that are thought to act by recruiting the RNA polymerase II holoenzyme to DNA in the absence of a classic activating region. These hybrid proteins (e.g., Gal11+Pho4 or Gal4(58-97)+Pho4 in the presence of a GAL11P allele) efficiently activated transcription and remodeled chromatin. Similar chromatin remodeling was observed at a PHO5 promoter deleted for TATA and thus unable to support transcription. We conclude that recruitment of the holoenzyme or associated proteins suffices for chromatin remodeling. We also show that the SWI/SNF complex is required neither for efficient transcription of the wild-type PHO5 nor the GAL1 promoters, and we observe nearly complete chromatin remodeling at PHO5 in the absence of Snf2.

Transcription factors vs nucleosomes: regulation of the PHO5 promoter in yeast.

Activation of the Saccharomyces cerevisiae PHO5 gene is accompanied by the disruption of four positioned nucleosomes at the promoter. The chromatin transition requires a DNA-binding protein, Pho4, and its transactivation domain. The mechanism of nucleosome disruption and the contribution of the nucleosomes to PHO5 regulation are reviewed.

The homeodomain protein Pho2 and the basic-helix-loop-helix protein Pho4 bind DNA cooperatively at the yeast PHO5 promoter.

Two transcription factors, the bHLH protein Pho4 and the homeodomain protein Pho2, are required for transcriptional activation of the PHO5 promoter in Saccharomyces cerevisiae. There are two essential Pho4 binding sites, corresponding to the regulatory elements UASp1 and UASp2 at the PHO5 promoter, but only a single, dispensable Pho2 binding site had previously been identified. We have reinvestigated binding of Pho2 to the PHO5 promoter using purified recombinant protein and have found multiple Pho2 binding sites of different affinities along the promoter. One of the high affinity Pho2 sites largely overlaps the Pho4 binding site at UASp1. Cooperative DNA binding of the two proteins to their overlapping sites, resulting in a high-affinity ternary complex, was demonstrated. Pho2 and Pho4 also bind DNA cooperatively at UASp2 where two Pho2 sites flank the Pho4 site. Finally, Pho2 facilitates binding of Pho4 to a third, cryptic Pho4 binding site which binds Pho4 with lower affinity than UASp1 or UASp2. These results suggest that cooperative DNA binding with Pho4 is integral to the mechanism by which Pho2 regulates transcription of the PHO5 gene.

Regulation of gene expression by nucleosomes.

During the past year, the characterization of mechanisms and factors capable of disrupting nucleosomes during transcriptional activation has been a recurrent theme in studies which address the contribution of nucleosome structure to gene regulation. In vivo studies using yeast and Drosophila together with biochemical purification schemes using nucleosome perturbation assays have provided evidence for the existence of multiprotein complexes that are able to alleviate nucleosome repression. At the same time, new insights into the mechanism of heterochromatin formation have been gained, which have direct links to nucleosome structure.

Interplay between nucleosomes and transcription factors at the yeast PHO5 promoter.

In this review, we summarize experiments which have used the yeast PHO5 gene to determine the functional consequences of nucleosome structure in the promoter region. In the PHO5 system, nucleosomes participate in promoter repression by interfering with factor binding. Therefore, disruption of nucleosome structure is likely a prerequisite for promoter activation. There still remain several important questions regarding the assembly and disassembly of chromatin repression. Recent experiments have shown that the PHO5 chromatin transition is replication and transcription independent. Nucleosome disruption does, however, depend upon binding of a transactivator, Pho4, to the PHO5 promoter. Moreover, the activation domain of Pho4 appears to play a critical role in chromatin disruption.

A nucleosome precludes binding of the transcription factor Pho4 in vivo to a critical target site in the PHO5 promoter.

Activation of the Saccharomyces cerevisiae PHO5 gene by phosphate starvation is accompanied by the disappearance of two pairs of positioned nucleosomes that flank a short hypersensitive region in the promoter. The transcription factor Pho4 is the key regulator of this transition. By in vitro footprinting it was previously shown that there is a low affinity site (UASp1) which is contained in the short hypersensitive region in the inactive promoter, and a high affinity site (UASp2) which is located in the adjacent nucleosome. To investigate the interplay between nucleosomes and Pho4, we have performed in vivo footprinting experiments with dimethylsulfate. Pho4 was found to bind to both sites in the active promoter. In contrast, it binds to neither site in the repressed promoter. Lack of binding under repressing conditions is largely due to the low affinity of Pho4 for its binding sites under these conditions. Despite the increased affinity of Pho4 for its target sites under activating conditions, binding to UASp2 is prevented by the presence of the nucleosome and can only occur after prior disruption of this nucleosome in a process that requires UASp1. Protection of the PHO5 UASp2 by the nucleosome is not absolute, however, since overexpression of Pho4 can disrupt this nucleosome even when UASp1 is deleted. Also under these conditions, with only UASp2 present, all four nucleosomes at the PHO5 promoter are disrupted, whereas no chromatin change at all is observed when both UAS elements are destroyed.