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.

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 ().

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.