H2A N-terminal Tail Research Articles

Mechanism of nucleosomal H2A K13/15 monoubiquitination and adjacent dual monoubiquitination by RNF168.

The DNA damage repair regulatory protein RNF168, a monomeric RING-type E3 ligase, has a crucial role in regulating cell fate and DNA repair by specific and efficient ubiquitination of the adjacent K13 and K15 (K13/15) sites at the H2A N-terminal tail. However, understanding how RNF168 coordinates with its cognate E2 enzyme UbcH5c to site-specifically ubiquitinate H2A K13/15 has long been hampered by the lack of high-resolution structures of RNF168 and UbcH5c~Ub (ubiquitin) in complex with nucleosomes. Here we developed chemical strategies and determined the cryo-electron microscopy structures of the RNF168-UbcH5c~Ub-nucleosome complex captured in transient H2A K13/15 monoubiquitination and adjacent dual monoubiquitination reactions, providing a 'helix-anchoring' mode for monomeric E3 ligase RNF168 on nucleosome in contrast to the 'compass-binding' mode of dimeric E3 ligases. Our work not only provides structural snapshots of H2A K13/15 site-specific monoubiquitination and adjacent dual monoubiquitination but also offers a near-atomic-resolution structural framework for understanding pathogenic amino acid substitutions and physiological modifications of RNF168.

Histone protein surface accessibility dictates direction of RSC-dependent nucleosome mobilization.

Chromatin remodeling enzymes use energy derived from ATP hydrolysis to mobilize nucleosomes and alter their structure to facilitate DNA access. The Remodels the Structure of Chromatin (RSC) complex has been extensively studied, yet aspects of how this complex functionally interacts with nucleosomes remain unclear. We introduce a steric mapping approach to determine how RSC activity depends on interaction with specific surfaces within the nucleosome. We find that blocking SHL + 4.5/–4.5 via streptavidin binding to the H2A N-terminal tail domains results in inhibition of RSC nucleosome mobilization. However, restriction enzyme assays indicate that remodeling-dependent exposure of an internal DNA site near the nucleosome dyad is not affected. In contrast, occlusion of both protein faces of the nucleosome by streptavidin attachment near the acidic patch completely blocks both remodeling-dependent nucleosome mobilization and internal DNA site exposure. However, we observed partial inhibition when only one protein surface is occluded, consistent with abrogation of one of two productive RSC binding orientations. Our results indicate that nucleosome mobilization requires RSC access to the trailing but not the leading protein surface, and reveals a mechanism by which RSC and related complexes may drive unidirectional movement of nucleosomes to regulate cis-acting DNA sequences in vivo.

DNA Damage-Induced Phosphorylation of Histone H2A at Serine 15 Is Linked to DNA End Resection.

The repair of DNA double-strand breaks (DSBs) occurs in chromatin, and several histone posttranslational modifications have been implicated in the process. Modifications of the histone H2A N-terminal tail have also been linked to DNA damage response, through acetylation or ubiquitination of lysine residues that regulate repair pathway choice. Here, we characterize a new DNA damage-induced phosphorylation on chromatin, at serine 15 of H2A in yeast. We show that this SQ motif functions independently of the classical S129 C-terminal site (γ-H2A) and that mutant-mimicking constitutive phosphorylation increases cell sensitivity to DNA damage. H2AS129ph is induced by Tel1ATM and Mec1ATR, and the loss of Lcd1ATRIP or Mec1 signaling decreases γ-H2A spreading distal to the DSB. In contrast, H2AS15ph is completely dependent on Lcd1ATRIP, indicating that this modification only happens when end resection is engaged. This is supported by an increase in replication protein A (RPA) and a decrease in DNA signal near the DSB in H2A-S15E phosphomimic mutants, indicating higher resection. In mammals, this serine is replaced by a lysine (H2AK15) which undergoes an acetyl-monoubiquityl switch to regulate binding of 53BP1 and resection. This regulation seems functionally conserved with budding yeast H2AS15 and 53BP1-homolog Rad9, using different posttranslational modifications between organisms but achieving the same function.

The role of histone tails in nucleosome stability: An electrostatic perspective

We propose a methodology for the study of protein-DNA electrostatic interactions and apply it to clarify the effect of histone tails in nucleosomes. This method can be used to correlate electrostatic interactions to structural and functional features of protein-DNA systems, and can be combined with coarse-grained representations. In particular, we focus on the electrostatic field and resulting forces acting on the DNA. We investigate the electrostatic origins of effects such as different stages in DNA unwrapping, nucleosome destabilization upon histone tail truncation, and the role of specific arginines and lysines undergoing Post-Translational Modifications. We find that the positioning of the histone tails can oppose the attractive pull of the histone core, locally deform the DNA, and tune DNA unwrapping. Small conformational variations in the often overlooked H2A C-terminal tails had significant electrostatic repercussions near the DNA entry and exit sites. The H2A N-terminal tail exerts attractive electrostatic forces towards the histone core in positions where Polymerase II halts its progress. We validate our results with comparisons to previous experimental and computational observations.

Dynamics of Nucleosome Tails Studied by All-Atom and Coarse-Grained MD Simulations

DNA in nucleosomes is sterically occluded and nucleosomes must open to allow full DNA access. This process is significantly influenced by the histone tails. Thus, here we studied the effect of histone tail modifications by all-atom MD simulations with 100 ns trajectories of the full nucleosome. We find evidence for an internal allosteric transition at the H2A-H3 interface induced by the removal of either the H3 or the H2A N-terminal tail. DNA dynamics on the histone core are studied by a new coarse-grained model; these simulations show a strong influence of the presence of the histone tails on DNA unwrapping, and an open state that can be stabilized by the displacement of the H3 tail into the gap between the DNA and the histone core.

Nucleosome Dynamics Studied by Single-Pair FRET and Computer Simulations

DNA in nucleosomes is sterically occluded and nucleosomes must open to allow full DNA access. We studied this process by single pair FRET (spFRET), all-atom and coarse-grained molecular dynamics. spFRET shows evidence for a new structural intermediate preceding histone dissociation, in which the (H3-H4)2 tetramer/(H2A-H2B) dimer interface is split open. This is followed by H2A-H2B dimer release from the DNA and, lastly, (H3-H4)2 tetramer removal. This open intermediate state could be demonstrated in Xenopus and yeast nucleosomes; histone variants such as H2A.Z change the mechanism of opening. We estimate that the open state is populated at 0.2 - 3 % under physiological conditions, and could have significant in vivo implications for factor-mediated histone exchange and for DNA accessibility. To look for subpopulations and changes in FRET distances during the salt-induced transition for the various FRET pairs, we used photon distribution analysis (PDA). We observed two to three populations for all the FRET pairs, their calculated distance corresponding to the values expected from the dye positions in the crystal structure. Histone tail acetylation increased these distances and shifted the midpoint of the opening transition to lower salt concentrations. The effect of histone tail modification was studied by all-atom MD simulations with 100 ns trajectories of the full nucleosome. We find evidence for an internal allosteric transition at the H2A-H3 interface induced by the removal of either the H3 or the H2A N-terminal tail. DNA dynamics on the histone core are studied by a new coarse-grained model; these simulations show a strong influence of the presence of the histone tails on DNA unwrapping, and an open state that can be stabilized by the displacement of the H3 tail into the gap between the DNA and the histone core.

The histone H2A N‐terminal tail regulates H2B monoubiquitylation and H3 K4 methylation via a novel trans tail pathway

The roles of the histone H3 and H4 N‐terminal tails in regulating chromatin structure and gene transcription have been extensively studied. However, very little is known about the functions of the H2A N‐terminal tail in transcription regulation. We analyzed multiple histone modifications associated with active transcription in the absence of the histone H2A tail. Our results reveal a novel trans‐tail regulation between the H2A tail and H2BK123 monoubiquitylation (H2BK123ub). Deletion of the H2A tail, or as little as four amino acids (a.a. 16–20), leads to a significant decrease in H2BK123ub, followed by a global reduction in H3K4 methylation by COMPASS. COMPASS complex purified from a H2A tail deletion strain lacks the Cps35 subunit, similar to the complex purified from a RAD6 deletion mutant. Rad6, which is responsible for ubiquitylating H2B K123, is still capable of being targeted to genes in the absence of the H2A tail, suggesting a type of post‐recruitment regulation. Deletion of the H2A tail causes a lag in the induction time of the GAL1 gene compared to wild type. Additionally, we show that deleting the Ubp8 and Ubp10 deubiquitylases not only restores the normal rate of induction of GAL1, but also restores H2BK123ub and H3K4me3 levels within the cell. Our analysis uncovers a role for the H2A tail in regulating transcription by maintaining a proper level of H2BK123 ubiquitylation and H3K4 methylation.This research was supported by funds from National Institutes of Health (GM58672) to J.C.R.

Regulation of Gene Transcription by the Histone H2A N-Terminal Domain

Histone N-terminal domains play critical roles in regulating chromatin structure and gene transcription. Relatively little is known, however, about the role of the histone H2A N-terminal domain in transcription regulation. We have used DNA microarrays to characterize the changes in genome-wide expression caused by mutations in the N-terminal domain of histone H2A. Our results indicate that the N-terminal domain of histone H2A functions primarily to repress the transcription of a large subset of the Saccharomyces cerevisiae genome and that most of the H2A-repressed genes are also repressed by the histone H2B N-terminal domain. Using the histone H2A microarray data, we selected three reporter genes (BNA1, BNA2, and GCY1), which we subsequently used to map regions in the H2A N-terminal domain responsible for this transcriptional repression. These studies revealed that a small subdomain in the H2A N-terminal tail, comprised of residues 16 to 20, is required for the transcriptional repression of these reporter genes. Deletion of either the entire histone H2A N-terminal domain or just this small subdomain imparts sensitivity to UV irradiation. Finally, we show that two residues in this H2A subdomain, serine-17 and arginine-18, are specifically required for the transcriptional repression of the BNA2 reporter gene.

Structural and functional conservation of the NuA4 histone acetyltransferase complex from yeast to humans.

The NuA4 histone acetyltransferase (HAT) multisubunit complex is responsible for acetylation of histone H4 and H2A N-terminal tails in yeast. Its catalytic component, Esa1, is essential for cell cycle progression, gene-specific regulation and has been implicated in DNA repair. Almost all NuA4 subunits have clear homologues in higher eukaryotes, suggesting that the complex is conserved throughout evolution to metazoans. We demonstrate here that NuA4 complexes are indeed present in human cells. Tip60 and its splice variant Tip60b/PLIP were purified as stable HAT complexes associated with identical polypeptides, with 11 of the 12 proteins being homologs of yeast NuA4 subunits. This indicates a highly conserved subunit composition and the identified human proteins underline the role of NuA4 in the control of mammalian cell proliferation. ING3, a member of the ING family of growth regulators, links NuA4 to p53 function which we confirmed in vivo. Proteins specific to the human NuA4 complexes include ruvB-like helicases and a bromodomain-containing subunit linked to ligand-dependent transcription activation by the thyroid hormone receptor. We also demonstrate that subunits MRG15 and DMAP1 are present in distinct protein complexes harboring histone deacetylase and SWI2-related ATPase activities, respectively. Finally, analogous to yeast, a recombinant trimeric complex formed by Tip60, EPC1, and ING3 is sufficient to reconstitute robust nucleosomal HAT activity in vitro. In conclusion, the NuA4 HAT complex is highly conserved in eukaryotes, in which it plays primary roles in transcription, cellular response to DNA damage, and cell cycle control.

The nonessential H2A N-terminal tail can function as an essential charge patch on the H2A.Z variant N-terminal tail.

Tetrahymena thermophila cells contain three forms of H2A: major H2A.1 and H2A.2, which make up approximately 80% of total H2A, and a conserved variant, H2A.Z. We showed previously that acetylation of H2A.Z was essential (Q. Ren and M. A. Gorovsky, Mol. Cell 7:1329-1335, 2001). Here we used in vitro mutagenesis of lysine residues, coupled with gene replacement, to identify the sites of acetylation of the N-terminal tail of the major H2A and to analyze its function in vivo. Tetrahymena cells survived with all five acetylatable lysines replaced by arginines plus a mutation that abolished acetylation of the N-terminal serine normally found in the wild-type protein. Thus, neither posttranslational nor cotranslational acetylation of major H2A is essential. Surprisingly, the nonacetylatable N-terminal tail of the major H2A was able to replace the essential function of the acetylation of the H2A.Z N-terminal tail. Tail-swapping experiments between H2A.1 and H2A.Z revealed that the nonessential acetylation of the major H2A N-terminal tail can be made to function as an essential charge patch in place of the H2A.Z N-terminal tail and that while the pattern of acetylation of an H2A N-terminal tail is determined by the tail sequence, the effects of acetylation on viability are determined by properties of the H2A core and not those of the N-terminal tail itself.

HSWI/SNF disrupts interactions between the H2A N-terminal tail and nucleosomal DNA.

We have employed a site-specific core histone-DNA cross-linking approach to investigate the mechanism of hSWI/SNF remodeling of a nucleosome. Remodeling results in the complete loss of canonical contacts between the N-terminal tail of H2A and DNA while new interactions are detected between this domain and DNA near the center of the original nucleosome. The data are consistent with a model in which remodeling results in the unraveling of a region of DNA from the edge of the nucleosome, leading to a repositioning of the H2A/H2B dimer to a noncanonical position near the center of the remodeled complex. Additionally, we find that prior cross-linking of the H2A N-terminal region to nucleosomal DNA does not restrict hSWI/SNF remodeling of the remainder of the nucleosome. Thus, disruption of both H2A-DNA interactions near the edge of the nucleosome is not an obligatory step in remodeling of the remainder of the complex.

Histone–Tryptase Interaction: H2A N-Terminal Tail Removal and Inhibitory Activity

The involvement of tryptase, the trypsin-like serine proteinase of mast cell granules, in many (patho)physiological conditions is now recognized.In vitrothis enzyme is known to act as a potent growth factor for fibroblasts and epithelial cells. Moreover, a role in inflammatory diseases and in dermatological disorders characterized by increased cell turnover has been suggested for this protease. In an attempt to understand the molecular basis of tryptase activity, we have investigated the interactionin vitrobetween bovine tryptase and histones. Here we show that tryptase cleaves histone H2A at a specific site (Arg20–Ala21), resulting in the removal of the N-terminal flexible fragment of the molecule. Furthermore, we demonstrate that the H2A major fragment (H2A*, 109 residues) generated by hydrolysis and lacking the N-terminal domain, is a noncompetitive, reversible and highly specific inhibitor (Ki= 29 nM) of tryptase enzymatic activity. H2A* is able to inhibit the hydrolysis of a small substrate as well as the cleavage of fibronectin, a high-molecular-weight substrate of tryptase.