Lessons from genome-wide studies: an integrated definition of the coactivator function of histone acetyl transferases
- Krishanpal Anamika†1, 2,
- Arnaud R Krebs†1,
- Julie Thompson2,
- Olivier Poch2,
- Didier Devys1 and
- Làszlò Tora1Email author
© Anamika et al; licensee BioMed Central Ltd. 2010
Received: 16 July 2010
Accepted: 20 October 2010
Published: 20 October 2010
Histone acetylation is one of the key regulatory mechanisms controlling transcriptional activity in eukaryotic cells. In higher eukaryotes, a number of nuclear histone acetyltransferase (HAT) enzymes have been identified, most of which are part of a large multisubunit complex. This diversity, combined with the large number of potentially acetylable lysines on histones, suggested the existence of a specific regulatory mechanism based on the substrate specificity of HATs. Over the past decade, intensive characterisations of the HAT complexes have been carried out. However, the precise mode of action of HATs, and particularly the functional differences amongst these complexes, remains elusive. Here we review current insights into the functional role of HATs, focusing on the specificity of their action. Studies based on biochemical as well as genetic approaches suggested that HATs exert a high degree of specificity in their acetylation spectra and in the cellular processes they regulate. However, a different view emerged recently from genomic approaches that provided genome-wide maps of HAT recruitments. The careful analysis of genomic data suggests that all HAT complexes would be simultaneously recruited to a similar set of loci in the genome, arguing for a low specificity in their function. In this review, we discuss the significance of these apparent contradictions and suggest a new model that integrates biochemical, genetic and genome-wide data to better describe the functional specificity of HAT complexes.
Histone post-translational modifications have shown to be key regulators among transcription regulation mechanisms [1, 2]. Histone acetylation is known to play an important role in the regulation of transcriptional activity in eukaryotic cells  by affecting higher-order folding of chromatin fibres, loosening of the contacts between the DNA and the nucleosomes and/or histone-nonhistone protein interactions [4–8]. Histone acetylation on various target lysines is in general positively associated with gene expression. Thus, HATs (also called lysine acetyl transferases or KATs) are thought to increase the decompaction of chromatin, which in turn may increase the accessibility of factors that promote transcription [8–11]. In higher eukaryotes, two enzymatic families (GNAT and MYST), each containing a dozen histone acetyltransferase (HAT) enzymes, have been identified and have often been shown to be subunits of larger transcriptional coactivator complexes.
Over the past decade, two approaches were mainly used to better understand the functional specificity of HATs. First, in vitro acetylation assays were carried out to investigate the substrate specificity of distinct HATs. These analyses showed that HATs exert a certain degree of specificity for particular lysine residues on different histone tails. Second, in vivo gene inactivation studies allowed testing the HAT specificity by observing phenotypical effects caused by ablation of a particular HAT. Interestingly, most of these studies argued for a high degree of specificity in the developmental or gene expression phenotypes. More recently, availability of new high-throughput technologies such as chromatin immunoprecipitation sequencing (ChIP-seq) allowed the investigation of the recruitment of HATs and the deposition of acetylation marks at a genome-wide scale [12, 13]. Contrary to the biochemical and genetic evidence, when carefully analysed, the genome-wide data suggest a low specificity in the recruitment and activity of HATs.
Here we comparatively review the conclusions of the above-mentioned three different approaches. Additionally, we discuss the significance of the conclusions made from each approach and try to reconcile new genome-wide evidence with existing knowledge in the form of a new model for the mode of action of HATs in transcriptional activation.
Biochemistry: Each HAT has a specific histone acetylation spectrum modulated by its macromolecular complex
Genetics: HATs exert a high degree of functional specificity
A remarkable feature emerging from sequence alignment of vertebrate HATs is the presence of highly related paralogs [i.e., CBP (CREB-binding protein, KAT3A)/p300 (KAT3B) or Gcn5/PCAF], whereas for most of the nuclear HATs only a single gene is present in nonvertebrate species. This observation raised the question of the functional importance of closely related paralogs in vertebrates. Answers were partially obtained by crossing mice carrying individual homozygous or heterozygous KOs of the given paralogs and comparing double KOs to that of the single KOs. For example, while the single CBP or p300 heterozygous mutants exert a milder phenotype than the homozygous mutants, the mice heterozygous for both HATs show phenotypes similar to the homozygous depletion of one or the other paralog . This result suggests that there is a functional redundancy between these paralogs and that in certain cases the dose of expression of the two paralogs is crucial for the proper development of the animals.
Taken together, these studies suggest that, amongst HATs, a high degree of functional specificity exists, with the exception of closely related paralogs that rather reflect the importance of gene expression dosage than functional specificity.
Genome-wide mapping: HATs are co-occurring at high frequency, creating hyperacetylated environment
In the past few years, our understanding of genome regulation has tremendously progressed due to the introduction of ChIP combined with microarray (ChIP-chip) or with high-throughput sequencing (ChIP-Seq). These experiments have allowed us to analyse the presence of a particular genomic feature at the genome-wide scale. Recently, several systematic ChIP-seq studies have been published, providing genome-wide mapping data for HATs and acetylation marks in resting human T cells [12, 13].
The detected co-occurrence of HATs and the consequent broad acetylation patterns observed can suggest two different scenarios. First, since in ChIP cell populations rather than a single cell are analysed, we cannot exclude that the recruited HATs could occur in a stochastic manner. This would imply that in a given cell, one HAT is recruited to a particular locus, while in another cell, a distinct HAT is recruited instead to the same locus. In this scenario, the observed lack of histone acetylation specificity would come from the lack of specificity in the mechanisms that recruit HATs to particular loci. In this case, the acetylation specificity would not be an important feature for regulation, but only the acetylation per se would be requested for proper activation.
In the second scenario, HATs would work collaboratively. In all cells, all the studied HATs would be permanently recruited and released at every bound loci in a dynamic fashion. This scenario implies also that many transcriptional coactivators can be dynamically recruited all the time to a set of regulated loci to modify (acetylate) the given chromatin environment. Such a mechanism for coactivator action has already been suggested by Hager et al. . Thus, gene regulation would mainly rely on the local abundance of the different coactivators rather than on the recruitment of a specific coactivator and its corresponding specific pattern of histone modifications. In this case, there would be a collaborative effect of all the HATs on the deposition of histone acetylation marks at the regulated set of genes.
An integrated model depicting mode of action of HATs
The genome-wide results do not directly contradict the biochemical evidence, since the broad in vivo co-occurrence of HAT enzymes at many genomic loci would explain the observation of multilysine hyperacetylated loci in vivo. However, this model is challenged by the conclusions of the genetic studies. It is clear from the KO studies that different HATs cannot compensate for each other's function. Moreover, if each process would require all the HATs, one would expect that all HAT KOs would result in the same phenotype, since all HAT-dependent processes should be affected. Thus a model assuming a complete overlap in HAT function can be excluded, and new hypotheses have to be raised to integrate the new lines of genome-wide evidence.
The time is a variable that was not introduced in the previous formulation of hypotheses. The analysed ChIP-seq data were all produced at a time t that reflects a single stage of the transcription activation process. Since the analysed genome-wide data were generated in resting somatic cells (CD4+ T cells) that are not challenged, one can assume that they exert only "routine" gene expression programs compared to the major transcriptional changes happening during differentiation of a tissue or an organism. We hypothesize that the genome-wide data obtained in the resting somatic cells reflect a "maintenance stage" in the gene activation process (Figure 5c). In our novel model, we propose that HATs could have a dual mode of action by distinguishing "initiation" and "maintenance" stages during the transcription activation process. The "initiation" stage would reflect the initial steps in the switch from an inactive to active transcription state, while the "maintenance" stage would represent the stabilisation of an active transcription stage over the time. In the initiation stage, the transcriptional activation of a given gene would be highly dependent on the specific recruitment of one given HAT (Figure 5c, left). Following this "initiation" stage, through a sequential process, this initial activation would lead to the consequent recruitment or binding of multiple HATs, which may recognize the initially open and acetylated environment in a less specific manner. This hypothesis is in good agreement with the findings that many HAT-containing complexes contain subunits with bromodomains that are thought to bind to acetylated histone tails. The binding of several HATs to the initially acetylated locus would thus serve to maintain the acetylation level and by consequence the activation state of a given locus (Figure 5c, right).
Our model would thus explain why in dynamic developmental processes HATs show high functional specificity (genetic data) that may not be the case in resting differentiated somatic cells. During cell differentiation, the high complexity level of transcriptional regulatory networks is a prerequisite for the orchestration of the time-controlled initiation of the proper transcriptional programs. However, in resting somatic tissues, where the number of newly initiated transcription processes is likely to be lower than during cell commitment, the large panel of activators would redundantly maintain activation states. This could explain why high redundancy in the recruitment of HATs is observed in resting cells, while their functional specificity is needed to achieve proper embryo development.
The above-presented data  is focused on catalytic subunits of HAT complexes. However, as discussed previously, a given HAT enzyme is (with the notable exception of CBP/p300) often embedded in more than one large multisubunit complex. Therefore, although the distinct HAT enzymes appear to be corecruited at many loci, they can represent the occurrence of different HAT complexes. Indeed, two recent studies analysed the differences of the recruitment between the two MOF-containing complexes (called NLS and MLS) at the genome-wide scale in Drosophila [24, 25]. The studies, by targeting NLS- or MLS-specific subunits, show a high specificity in the recruitment of these complexes. Thus, by studying individually the well-defined HAT complexes, the resolution of the analysis will increase, leading to a more precise separation of the observed overlap in the recruitment of the HATs. Nevertheless, the existence of specific loci, where a single HAT is recruited should not be excluded. However, according to the analysis of the data from the Zhao lab , these events appear to be the exception rather than the rule.
The very recent improvement of the genome-wide mapping technologies and the development of novel ChIP-grade antibodies against HATs and complex-specific subunits will allow researchers to verify these genome-wide observations and provide new insights into the functional roles of HATs. For example, the time course analysis of different HAT recruitments upon dynamic activation of a particular gene networks, for example, in vitro stem cell differentiation systems , should allow researchers to test the proposed model.
HAT-containing complexes are key components of chromatin-mediated transcriptional regulatory networks. Proper understanding and modelling of their mode of action and function within these networks is a prerequisite for accurate prediction of the behaviour of transcriptional systems. Recent technology breakthrough in the postgenomic area allowed new insights into the function of HATs, but were often interpreted with the liability of ignoring previously available biochemical and genetic data, leading to oversimplified models. Here we propose novel considerations that could reconcile the different lines of evidence in a unified model that describes the mode of action of HATs more precisely. Our current model proposes that HATs are differently required, depending on the stage of gene activation, with a high functional specificity in the early gene activation stage and with a less-specific functionality in the later maintenance stage.
We thank S. Bour for artwork and T. Ye for bioinformatics support. We apologize to colleagues, whose work was only covered by reviews. ARK is recipient of a fellowship from INSERM-Région Alsace and the Association pour la Recherche sur le Cancer (ARC). This work was funded by grants from ANR (GenomATAC; ANR-09-BLAN-0266) and the EU (EUTRACC, LSHG-CT-2007-037445).
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