Mechanisms of stimulation of SAGA-mediated nucleosome acetylation by a transcriptional activator.

Eukaryotic gene expression requires the coordination of multiple factors to overcome the repressive nature of chromatin. However, the mechanistic details of this coordination are not well understood. The SAGA family of transcriptional coactivators interacts with DNA-binding activators to establish regions of hyperacetylation. We have previously shown that, contrary to the prevailing model in which activator protein increases SAGA affinity for nucleosome substrate, the Gal4-VP16 activator model system augments the rate of acetylation turnover for the SAGA complex from budding yeast. To better understand how this stimulation occurs, we have identified necessary components using both kinetics assays and binding interactions studies. We find that Gal4-VP16-mediated stimulation requires activator binding to DNA flanking the nucleosome, as it cannot be reproduced in trans by activator protein alone or by exogenous DNA containing the activator binding site in combination with the activator protein. Further, activator-mediated stimulation requires subunits outside of the histone acetylation (HAT) module, with the Tra1 subunit being responsible for the majority of the stimulation. Interestingly, for the HAT module alone, nucleosome acetylation is inhibited by activator proteins due to non-specific binding of the activator to the nucleosomes. This inhibition is not observed for the yeast ADA complex, a small complex comprised mostly of the HAT module, suggesting that subunits outside of the HAT module in both it and SAGA can overcome non-specific activator binding to nucleosomes. However, this activity appears distinct from activator-mediated stimulation, as ADA complex acetylation is not stimulated by Gal4-VP16.

Distinct requirements of linker DNA and transcriptional activators in promoting SAGA-mediated nucleosome acetylation.

The Spt-Ada-Gcn5 acetyltransferase (SAGA) family of transcriptional coactivators are prototypical nucleosome acetyltransferase complexes that regulate multiple steps in gene transcription. The size and complexity of both the SAGA enzyme and the chromatin substrate provide numerous opportunities for regulating the acetylation process. To better probe this regulation, here we developed a bead-based nucleosome acetylation assay to characterize the binding interactions and kinetics of acetylation with different nucleosomal substrates and the full SAGA complex purified from budding yeast (Saccharomyces cerevisiae). We found that SAGA-mediated nucleosome acetylation is stimulated up to 9-fold by DNA flanking the nucleosome, both by facilitating the binding of SAGA and by accelerating acetylation turnover. This stimulation required that flanking DNA is present on both sides of the nucleosome and that one side is >15 bp long. The Gal4-VP16 transcriptional activator fusion protein could also augment nucleosome acetylation up to 5-fold. However, contrary to our expectations, this stimulation did not appear to occur by stabilizing the binding of SAGA toward nucleosomes containing an activator-binding site. Instead, increased acetylation turnover by SAGA stimulated nucleosome acetylation. These results suggest that the Gal4-VP16 transcriptional activator directly stimulates acetylation via a dual interaction with both flanking DNA and SAGA. Altogether, these findings uncover several critical mechanisms of SAGA regulation by chromatin substrates.

Structure, High Affinity, and Negative Cooperativity of the Escherichia coli Holo-(Acyl Carrier Protein):Holo-(Acyl Carrier Protein) Synthase Complex.

The Escherichia coli holo-(acyl carrier protein) synthase (ACPS) catalyzes the coenzyme A-dependent activation of apo-ACPP to generate holo-(acyl carrier protein) (holo-ACPP) in an early step of fatty acid biosynthesis. E. coli ACPS is sufficiently different from the human fatty acid synthase to justify the development of novel ACPS-targeting antibiotics. Models of E. coli ACPS in unliganded and holo-ACPP-bound forms solved by X-ray crystallography to 2.05and 4.10Å, respectively, revealed that ACPS bound three product holo-ACPP molecules to form a 3:3 hexamer. Solution NMR spectroscopy experiments validated the ACPS binding interface on holo-ACPP using chemical shift perturbations and by determining the relative orientation of holo-ACPP to ACPS by fitting residual dipolar couplings. The binding interface is organized to arrange contacts between positively charged ACPS residues and the holo-ACPP phosphopantetheine moiety, indicating product contains more stabilizing interactions than expected in the enzyme:substrate complex. Indeed, holo-ACPP bound the enzyme with greater affinity than the substrate, apo-ACPP, and with negative cooperativity. The first equivalent of holo-ACPP bound with a KD=62±13nM, followed by the binding of two more equivalents of holo-ACPP with KD=1.2±0.2µM. Cooperativity was not observed for apo-ACPP which bound with KD=2.4±0.1µM. Strong product binding and high levels of holo-ACPP in the cell identify a potential regulatory role of ACPS in fatty acid biosynthesis.

Solid-phase synthesis of highly repetitive chromatin assembly templates.

DNA templates for assembling chromatin model systems typically consist of numerous repeats of nucleosome positioning sequences, making their synthesis challenging. Here we describe a solid-phase strategy for generating such templates using sequential enzymatic ligation of DNA monomers. Using single nucleosome site monomers, we can either generate a twelve-nucleosome site target, or systematically access intermediate-sized templates. Using twelve nucleosome positioning site monomers, longer templates can be generated. Our synthesized templates assemble into well-defined chromatin model systems, demonstrating the utility of our solid-phase approach. Moreover, our strategy should be more widely applicable to generating other DNAs containing highly repetitive DNA sequences.

Expression and purification of histone H3 proteins containing multiple sites of lysine acetylation using nonsense suppression.

Lysine acetylation is a common post-translational modification, which is especially prevalent in histone proteins in chromatin. A number of strategies exist for generating histone proteins containing lysine acetylation, but an especially attractive approach is to genetically encode acetyl-lysine residues using nonsense suppression. This strategy has been successfully applied to single sites of histone acetylation. However, because histone acetylation can often occur at multiple sites simultaneously, we were interested in determining whether this approach could be extended. Here we show that we can express histone H3 proteins that incorporate up to four sites of lysine acetylation on the histone tail. Because the amount of expressed multi-acetylated histone is reduced relative to the wild type, a purification strategy involving affinity purification and ion exchange chromatography was optimized. This expression and purification strategy ultimately generates H3 histone uniformly acetylated at the desired position at levels and purity sufficient to assemble histone octamers. Histone octamers containing four sites of lysine acetylation were assembled into mononucleosomes and enzymatic assays confirmed that this acetylation largely blocks further acetylation by the yeast SAGA acetyltransferase complex.

Nucleosome acetylation sequencing to study the establishment of chromatin acetylation.

The establishment of posttranslational chromatin modifications is a major mechanism for regulating how genomic DNA is utilized. However, current in vitro chromatin assays do not monitor histone modifications at individual nucleosomes. Here we describe a strategy, nucleosome acetylation sequencing, that allows us to read the amount of modification at each nucleosome. In this approach, a bead-bound trinucleosome substrate is enzymatically acetylated with radiolabeled acetyl CoA by the SAGA complex from Saccharomyces cerevisae. The product is digested by restriction enzymes that cut at unique sites between the nucleosomes and then counted to quantify the extent of acetylation at each nucleosomal site. We find that we can sensitively, specifically, and reproducibly follow enzyme-mediated nucleosome acetylation. Applying this strategy, when acetylation proceeds extensively, its distribution across nucleosomes is relatively uniform. However, when substrates are used that contain nucleosomes mutated at the major sites of SAGA-mediated acetylation, or that are studied under initial rate conditions, changes in the acetylation distribution can be observed. Nucleosome acetylation sequencing should be applicable to analyzing a wide range of modifications. Additionally, because our trinucleosomes synthesis strategy is highly modular and efficient, it can be used to generate nucleosomal systems in which nucleosome composition differs across the array.

Human heterochromatin protein 1α promotes nucleosome associations that drive chromatin condensation.

HP1(Hsα)-containing heterochromatin is located near centric regions of chromosomes and regulates DNA-mediated processes such as DNA repair and transcription. The higher-order structure of heterochromatin contributes to this regulation, yet the structure of heterochromatin is not well understood. We took a multidisciplinary approach to determine how HP1(Hsα)-nucleosome interactions contribute to the structure of heterochromatin. We show that HP1(Hsα) preferentially binds histone H3K9Me3-containing nucleosomal arrays in favor of non-methylated nucleosomal arrays and that nonspecific DNA interactions and pre-existing chromatin compaction promote binding. The chromo and chromo shadow domains of HP1(Hsα) play an essential role in HP1(Hsα)-nucleosome interactions, whereas the hinge region appears to have a less significant role. Electron microscopy of HP1(Hsα)-associated nucleosomal arrays showed that HP1(Hsα) caused nucleosome associations within an array, facilitating chromatin condensation. Differential sedimentation of HP1(Hsα)-associated nucleosomal arrays showed that HP1(Hsα) promotes interactions between arrays. These strand-to-strand interactions are supported by in vivo studies where tethering the Drosophila homologue HP1a to specific sites promotes interactions with distant chromosomal sites. Our findings demonstrate that HP1(Hsα)-nucleosome interactions cause chromatin condensation, a process that regulates many chromosome events.

Histone H3 tail acetylation modulates ATP-dependent remodeling through multiple mechanisms.

There is a close relationship between histone acetylation and ATP-dependent chromatin remodeling that is not fully understood. We show that acetylation of histone H3 tails affects SWI/SNF (mating type switching/ sucrose non fermenting) and RSC (remodels structure of chromatin) remodeling in several distinct ways. Acetylation of the histone H3 N-terminal tail facilitated recruitment and nucleosome mobilization by the ATP-dependent chromatin remodelers SWI/SNF and RSC. Tetra-acetylated H3, but not tetra-acetylated H4 tails, increased the affinity of RSC and SWI/SNF for nucleosomes while also changing the subunits of SWI/SNF that interact with the H3 tail. The enhanced recruitment of SWI/SNF due to H3 acetylation is bromodomain dependent, but is not further enhanced by additional bromodomains found in RSC. The combined effect of H3 acetylation and transcription activators is greater than either separately which suggests they act in parallel to recruit SWI/SNF. Besides enhancing recruitment, H3 acetylation increased nucleosome mobilization and H2A/H2B displacement by RSC and SWI/SNF in a bromodomain dependent manner and to a lesser extent enhanced ATP hydrolysis independent of bromodomains. H3 and H4 acetylation did not stimulate disassembly of adjacent nucleosomes in short arrays by SWI/SNF or RSC. These data illustrate how histone acetylation modulates RSC and SWI/SNF function, and provide a mechanistic insight into their collaborative efforts to remodel chromatin.

Nucleosome interactions and stability in an ordered nucleosome array model system.

Although it is well established that the majority of eukaryotic DNA is sequestered as nucleosomes, the higher-order structure resulting from nucleosome interactions as well as the dynamics of nucleosome stability are not as well understood. To characterize the structural and functional contribution of individual nucleosomal sites, we have developed a chromatin model system containing up to four nucleosomes, where the array composition, saturation, and length can be varied via the ordered ligation of distinct mononucleosomes. Using this system we find that the ligated tetranucleosomal arrays undergo intra-array compaction. However, this compaction is less extensive than for longer arrays and is histone H4 tail-independent, suggesting that well ordered stretches of four or fewer nucleosomes do not fully compact to the 30-nm fiber. Like longer arrays, the tetranucleosomal arrays exhibit cooperative self-association to form species composed of many copies of the array. This propensity for self-association decreases when the fraction of nucleosomes lacking H4 tails is systematically increased. However, even tetranucleosomal arrays with only two octamers possessing H4 tails recapitulate most of the inter-array self-association. Varying array length shows that systems as short as dinucleosomes demonstrate significant self-association, confirming that relatively few determinants are required for inter-array interactions and suggesting that in vivo multiple interactions of short runs of nucleosomes might contribute to complex fiber-fiber interactions. Additionally, we find that the stability of nucleosomes toward octamer loss increases with array length and saturation, suggesting that in vivo stretches of ordered, saturated nucleosomes could serve to protect these regions from histone ejection.

Role of direct interactions between the histone H4 Tail and the H2A core in long range nucleosome contacts.

In eukaryotic nuclei the majority of genomic DNA is believed to exist in higher order chromatin structures. Nonetheless, the nature of direct, long range nucleosome interactions that contribute to these structures is poorly understood. To determine whether these interactions are directly mediated by contacts between the histone H4 amino-terminal tail and the acidic patch of the H2A/H2B interface, as previously demonstrated for short range nucleosomal interactions, we have characterized the extent and effect of disulfide cross-linking between residues in histones contained in different strands of nucleosomal arrays. We show that in 208-12 5 S rDNA and 601-177-12 nucleosomal array systems, direct interactions between histones H4-V21C and H2A-E64C can be captured. This interaction depends on the extent of initial cross-strand association but does not require these specific residues, because interactions with residues flanking H4-V21C can also be captured. Additionally, we find that trapping H2A-H4 intra-array interactions antagonizes the ability of these arrays to undergo intermolecular self-association.

Recognition imaging of acetylated chromatin using a DNA aptamer.

Histone acetylation plays an important role in the regulation of gene expression. A DNA aptamer generated by in vitro selection to be highly specific for histone H4 protein acetylated at lysine 16 was used as a recognition element for atomic force microscopy-based recognition imaging of synthetic nucleosomal arrays with precisely controlled acetylation. The aptamer proved to be reasonably specific at recognizing acetylated histones, with recognition efficiencies of 60% on-target and 12% off-target. Though this selectivity is much poorer than the >2000:1 equilibrium specificity of the aptamer, it is a large improvement on the performance of a ChIP-quality antibody, which is not selective at all in this application, and it should permit high-fidelity recognition with repeated imaging. The ability to image the precise location of posttranslational modifications may permit nanometer-scale investigation of their effect on chromatin structure.

The Gcn5 bromodomain of the SAGA complex facilitates cooperative and cross-tail acetylation of nucleosomes.

Bromodomains are acetyl lysine binding modules found in many complexes that regulate gene transcription. In budding yeast, the coactivator complex SAGA (Spt-Ada-Gcn5-acetyl-transferase) predominantly facilitates transcription of stress-activated genes and requires the bromodomain of the Gcn5 subunit for full activation of a number of these genes. This bromodomain has previously been shown to promote retention of the complex to H3 and H4 acetylated nucleosomes. Because the SAGA complex mediates histone H3 acetylation, we sought to determine to what extent the Gcn5 bromodomain directly modulates histone acetylation activity. Kinetic analysis of SAGA-mediated acetylation of nucleosomal substrates reveals that this bromodomain: 1) is required for the cooperative acetylation of nucleosomes, 2) enhances acetylation of an H3 histone tail when the other H3 tail within a nucleosome is already acetylated, and 3) augments the acetylation turnover of nucleosomes previously acetylated at lysine 16 of the histone H4 tails. These results indicate that the Gcn5 bromodomain promotes the establishment of nucleosome acetylation through multiple mechanisms and more generally show how chromatin recognition domains can modulate the enzymatic activity of chromatin modifying complexes.

Cross-talk between histone H3 tails produces cooperative nucleosome acetylation.

Acetylation of histone proteins by the yeast Spt-Ada-Gcn5-acetyltansferase (SAGA) complex has served as a paradigm for understanding how posttranslational modifications of chromatin regulate eukaryotic gene expression. Nonetheless, it has been unclear to what extent the structural complexity of the chromatin substrate modulates SAGA activity. By using chromatin model systems, we have found that SAGA-mediated histone acetylation is highly cooperative (cooperativity constant of 1.97 +/- 0.15), employing the binding of multiple noncontiguous nucleosomes to facilitate maximal acetylation activity. Studies with various chromatin substrates, including those containing novel asymmetric histone octamers, indicate that this cooperativity occurs only when both H3 histone tails within a nucleosome are properly oriented and unacetylated. We propose that modulation of maximal SAGA activity through this dual-tail recognition could facilitate coregulation of spatially proximal genes by promoting cooperative nucleosome acetylation between genes.

Mimicking methylated histones.

Histones with specific patterns of lysine methylation help to define how their associated DNA is used. A recent semisynthetic strategy for generating histone proteins that contain methyl-lysine analogues at specific sites will provide researchers with the materials to further elucidate the role of these modifications.

Switching on chromatin: mechanistic role of histone H4-K16 acetylation.

How post-translational histone modifications regulate DNA utilization constitutes one of the central questions of chromatin biology. In studying the mechanistic role of histone H4-K16 acetylation, a mark with a functional role in maintaining transcriptionally permissive DNA domains or directly promoting gene transcription, we found that this acetylation both disrupts higher-order chromatin structure and changes the functional interaction of chromatin-associated proteins. The potential significance of this finding for in vivo chromatin structure, establishment of euchromatic domains, and promotion of gene transcription is examined.

Histone H4-K16 acetylation controls chromatin structure and protein interactions.

Acetylation of histone H4 on lysine 16 (H4-K16Ac) is a prevalent and reversible posttranslational chromatin modification in eukaryotes. To characterize the structural and functional role of this mark, we used a native chemical ligation strategy to generate histone H4 that was homogeneously acetylated at K16. The incorporation of this modified histone into nucleosomal arrays inhibits the formation of compact 30-nanometer-like fibers and impedes the ability of chromatin to form cross-fiber interactions. H4-K16Ac also inhibits the ability of the adenosine triphosphate-utilizing chromatin assembly and remodeling enzyme ACF to mobilize a mononucleosome, indicating that this single histone modification modulates both higher order chromatin structure and functional interactions between a nonhistone protein and the chromatin fiber.

A native peptide ligation strategy for deciphering nucleosomal histone modifications.

Post-translational modifications of histones influence both chromatin structure and the binding and function of chromatin-associated proteins. A major limitation to understanding these effects has been the inability to construct nucleosomes in vitro that harbor homogeneous and site-specific histone modifications. Here, we describe a native peptide ligation strategy for generating nucleosomal arrays that can harbor a wide range of desired histone modifications. As a first test of this method, we engineered model nucleosomal arrays in which each histone H3 contains a phosphorylated serine at position 10 and performed kinetic analyses of Gcn5-dependent histone acetyltransferase activities. Recombinant Gcn5 shows increased histone acetyltransferase activity on nucleosomal arrays harboring phosphorylated H3 serine 10 and is consistent with peptide studies. However, in contrast to analyses using peptide substrates, we find that the histone acetyltransferase activity of the Gcn5-containing SAGA complex is not stimulated by H3 phosphorylation in the context of nucleosomal arrays. This difference between peptide and array substrates suggests that the ability to generate specifically modified nucleosomal arrays should provide a powerful tool for understanding the effects of post-translational histone modifications.