Histone H2A-H2B Dimer Research Articles

To slide or not to slide: key role of the hexasome in chromatin remodeling revealed.

Hexasomes are non-canonical nucleosomes that package DNA with six instead of eight histones. First discovered 40 years ago as a consequence of transcription, two near-atomic-resolution cryo-EM structures of the hexasome in complex with the chromatin remodeler INO80 have now started to unravel its mechanistic impact on the regulatory landscape of chromatin. Loss of one histone H2A-H2B dimer converts inactive nucleosomes into distinct and favorable substrates for ATP-dependent chromatin remodeling.

Structural insights into histone exchange by human SRCAP complex

Histone variant H2A.Z is found at promoters and regulates transcription. The ATP-dependent chromatin remodeler SRCAP complex (SRCAP-C) promotes the replacement of canonical histone H2A–H2B dimer with H2A.Z–H2B dimer. Here, we determined structures of human SRCAP-C bound to H2A-containing nucleosome at near-atomic resolution. The SRCAP subunit integrates a 6-subunit actin-related protein (ARP) module and an ATPase-containing motor module. The ATPase-associated ARP module encircles half of the nucleosome along the DNA and may restrain net DNA translocation, a unique feature of SRCAP-C. The motor module adopts distinct nucleosome binding modes in the apo (nucleotide-free), ADP-bound, and ADP-BeFx-bound states, suggesting that ATPase-driven movement destabilizes H2A–H2B by unwrapping the entry DNA and pulls H2A–H2B out of nucleosome through the ZNHIT1 subunit. Structure-guided chromatin immunoprecipitation sequencing analysis confirmed the requirement of H2A-contacting ZNHIT1 in maintaining H2A.Z occupancy on the genome. Our study provides structural insights into the mechanism of H2A-H2A.Z exchange mediated by SRCAP-C.

Spontaneous histone exchange between nucleosomes

The nucleosome is the fundamental gene-packing unit in eukaryotes. Nucleosomes comprise ∼147bp DNA wrapped around an octameric histone protein core composed of two H2A-H2B dimers and one (H3-H4)2 tetramer. The strong yet flexible DNA-histone interactions are the physical basis of the dynamic regulation of genes packaged in chromatin. The dynamic nature of DNA-histone interactions also implies thatnucleosomes dissociate DNA-histone contacts both transiently and repeatedly. This kinetic instability may lead to spontaneous nucleosome disassembly or histone exchange between nucleosomes. At high nucleosome concentrations, nucleosome-nucleosome collisions and subsequent histone exchange would be a more likely event, where nucleosomes could act as their own histone chaperone. This spontaneous histone exchange could serve as a mechanism for maintaining overall chromatin stability, although it has never been reported. Here we employed three-color single-molecule FRET (smFRET) to demonstrate that histone H2A-H2B dimers are exchanged spontaneously between nucleosomes on a time scale of a few tens of seconds at a physiological nucleosome concentration. We show that the rate of histone exchange increases at a higher monovalent salt concentration, with histone-acetylated nucleosomes, and in the presence of histone chaperone Nap1, while it remains unchanged at a higher temperature, and decreases upon DNA methylation. These results support the notion of histone exchange via transient and repetitive partial disassembly of the nucleosome and corroborate spontaneous histone diffusion in a compact chromatin context, modulating the local concentrations of histone modifications and variants.

The histone H2B Arg95 residue efficiently recruits the transcription factor Spt16 to mediate Ste5 expression of the pheromone response pathway

In yeast Saccharomyces cerevisiae, the immunosuppressant rapamycin inhibits the TORC1 kinase causing rapid alteration in gene expression and leading to G1 arrest. We recently reported the isolation and characterization from the histone mutant collection of a histone H2B R95A mutant that displays resistance to rapamycin. This mutant is defective in the expression of several genes belonging to the pheromone response pathway including STE5 encoding a scaffold protein that promotes the activation of downstream MAP kinases. Cells lacking Ste5 cannot arrest the cell cycle in response to rapamycin and as a consequence exhibit similar resistance to rapamycin as the H2B R95A mutant. Herein, we show that the H2B R95A mutation weakens the association of H2B with Spt16 a component of the FACT complex (FAcilitates Chromatin Transcription), and an essential factor that interacts with the histone H2A-H2B dimer to promote transcription and preserve chromatin integrity. From a collection of spt16 mutants, spt16 E857K and spt16-11 showed striking sensitivity to rapamycin as compared to the parent strain. spt16 E857K and spt16-11 expressed distinct forms of Ste5, while a suppressor mutation H2B A84D of the spt16-11 mutant prevents the expression of Ste5 and confers marked resistance to rapamycin. We interpret these findings to suggest that the Arg95 residue of histone H2B is required to recruit Spt16 to maintain the expression of STE5, which performs a role to arrest cells in the G1 phase in response to rapamycin.

Cryo–electron microscopy structure of the H3-H4 octasome: A nucleosome-like particle without histones H2A and H2B

The canonical nucleosome, which represents the major packaging unit of eukaryotic chromatin, has an octameric core composed of two histone H2A-H2B and H3-H4 dimers with ∼147 base pairs (bp) of DNA wrapped around it. Non-nucleosomal particles with alternative histone stoichiometries and DNA wrapping configurations have been found, and they could profoundly influence genome architecture and function. Using cryo-electron microscopy, we solved the structure of the H3-H4 octasome, a nucleosome-like particle with a di-tetrameric core consisting exclusively of the H3 and H4 histones. The core is wrapped by ∼120 bp of DNA in 1.5 negative superhelical turns, forming two stacked disks that are connected by a H4-H4' four-helix bundle. Three conformations corresponding to alternative interdisk angles were observed, indicating the flexibility of the H3-H4 octasome structure. In vivo crosslinking experiments detected histone-histone interactions consistent with the H3-H4 octasome model, suggesting that H3-H4 octasomes or related structural features exist in cells.

Structure of human telomerase holoenzyme with bound telomeric DNA.

Telomerase adds telomeric repeats at chromosome ends to compensate for the telomere loss that is caused by incomplete genome end replication1. In humans, telomerase is upregulated during embryogenesis and in cancers, and mutations that compromise the function of telomerase result in disease2. A previous structure of human telomerase at a resolution of 8Å revealed a vertebrate-specific composition and architecture3, comprising a catalytic core that is flexibly tethered to an H and ACA (hereafter, H/ACA) box ribonucleoprotein (RNP) lobe by telomerase RNA. High-resolution structural information is necessary to develop treatments that can effectively modulate telomerase activity as a therapeutic approach against cancers and disease. Here we used cryo-electron microscopy to determine the structure of human telomerase holoenzyme bound to telomeric DNAat sub-4 Å resolution, which reveals crucial DNA- and RNA-binding interfaces in the active site of telomerase as well as the locations of mutations that alter telomerase activity. We identified a histone H2A-H2B dimer within the holoenzyme that was bound to an essential telomerase RNA motif, which suggests a role for histones in the folding and function of telomerase RNA. Furthermore, this structure of a eukaryotic H/ACA RNP reveals the molecular recognition of conserved RNA and protein motifs, as well as interactions that are crucial for understanding the molecular pathology of many mutations that cause disease. Our findings provide thestructural details of the assembly and active site of human telomerase, which paves the way for the development of therapeutic agents that target this enzyme.

COMMD4 functions with the histone H2A-H2B dimer for the timely repair of DNA double-strand breaks

Genomic stability is critical for normal cellular function and its deregulation is a universal hallmark of cancer. Here we outline a previously undescribed role of COMMD4 in maintaining genomic stability, by regulation of chromatin remodelling at sites of DNA double-strand breaks. At break-sites, COMMD4 binds to and protects histone H2B from monoubiquitination by RNF20/RNF40. DNA damage-induced phosphorylation of the H2A-H2B heterodimer disrupts the dimer allowing COMMD4 to preferentially bind H2A. Displacement of COMMD4 from H2B allows RNF20/40 to monoubiquitinate H2B and for remodelling of the break-site. Consistent with this critical function, COMMD4-deficient cells show excessive elongation of remodelled chromatin and failure of both non-homologous-end-joining and homologous recombination. We present peptide-mapping and mutagenesis data for the potential molecular mechanisms governing COMMD4-mediated chromatin regulation at DNA double-strand breaks.

COMMD4 Functions with the Histone H2A-H2B Dimer for the Timely Repair of DNA Double Strand Breaks

Genomic stability is critical for normal cellular function and its deregulation is a universal hallmark of cancer. Here we outline a previously undescribed role of COMMD4 in maintaining genomic stability, by regulating of chromatin remodelling at sites of DNA double strand breaks (DSBs). We demonstrate that COMMD4 is initially recruited to DSBs by a direct-interaction with phosphorylated hSSB1. At break-sites, COMMD4 binds to and protects histone H2B from monoubiquitination by RNF20/RNF40. DNA damage-induced phosphorylation of the H2A-H2B heterodimer disrupts the dimer allowing COMMD4 to preferentially bind H2A. Displacement of COMMD4 from H2B allows RNF20/40 to monoubuquitinate H2B and for remodelling of the break-site. Consistent with this critical function, COMMD4-defficient cells show excessive elongation of remodelled chromatin and failure of both non-homologous-end-joining and homologous recombination. We present a model, developed by molecular dynamic simulation, peptide-mapping and mutagenesis for the potential molecular mechanisms governing COMMD4-mediated chromatin regulation at DSBs.

Atomic resolution cryo-EM structure of a native-like CENP-A nucleosome aided by an antibody fragment

Genomic DNA in eukaryotes is organized into chromatin through association with core histones to form nucleosomes, each distinguished by their DNA sequences and histone variants. Here, we used a single-chain antibody fragment (scFv) derived from the anti-nucleosome antibody mAb PL2-6 to stabilize human CENP-A nucleosome containing a native α-satellite DNA and solved its structure by the cryo-electron microscopy (cryo-EM) to 2.6 Å resolution. In comparison, the corresponding cryo-EM structure of the free CENP-A nucleosome could only reach 3.4 Å resolution. We find that scFv binds to a conserved acidic patch on the histone H2A-H2B dimer without perturbing the nucleosome structure. Our results provide an atomic resolution cryo-EM structure of a nucleosome and insight into the structure and function of the CENP-A nucleosome. The scFv approach is applicable to the structural determination of other native-like nucleosomes with distinct DNA sequences.

DNA sequence influences hexasome orientation to regulate DNA accessibility.

Nucleosomes, the fundamental organizing units of eukaryotic genomes, contain ∼146 base pairs of DNA wrapped around a histone H3–H4 tetramer and two histone H2A–H2B dimers. Converting nucleosomes into hexasomes by removal of a H2A–H2B dimer is an important regulatory event, but its regulation and functional consequences are not well-understood. To investigate the influence of hexasomes on DNA accessibility, we used the property of the Widom-601 Nucleosome Positioning Sequence (NPS) to form homogeneously oriented hexasomes in vitro. We find that DNA accessibility to transcription factors (TF) on the hexasome H2A–H2B distal side is identical to naked DNA, while the accessibility on the H2A–H2B proximal side is reduced by 2-fold, which is due to a 2-fold reduction in hexasome unwrapping probability. We then determined that a 23 bp region of the Widom-601 NPS is responsible for forming homogeneously oriented hexasomes. Analysis of published ChIP-exo data of hexasome containing genes identified two DNA sequence motifs that correlate with hexasome orientation in vivo, while ExoIII mapping studies of these sequences revealed they generate homogeneously oriented hexasomes in vitro. These results indicate that hexasome orientation, which is influenced by the underlying DNA sequence in vivo, is important for modulating DNA accessibility to regulate transcription.

Functional roles of the DNA-binding HMGB domain in the histone chaperone FACT in nucleosome reorganization

The essential histone chaperone FACT (facilitates chromatin transcription) promotes both nucleosome assembly and disassembly. FACT is a heterodimer of Spt16 with either SSRP1 or Pob3, differing primarily by the presence of a high-mobility group B (HMGB) DNA-binding domain furnished only by SSRP1. Yeast FACT lacks the intrinsic HMGB domain found in SSRP1-based homologs such as human FACT, but yeast FACT activity is supported by Nhp6, which is a freestanding, single HMGB-domain protein. The importance of histone binding by FACT domains has been established, but the roles of DNA-binding activity remain poorly understood. Here, we examined these roles by fusing single or multiple HMGB modules to Pob3 to mimic SSRP1 or to test the effects of extended DNA-binding capacity. Human FACT and a yeast mimic both required Nhp6 to support nucleosome reorganization in vitro, indicating that a single intrinsic DNA-binding HMGB module is insufficient for full FACT activity. Three fused HMGB modules supported activity without Nhp6 assistance, but this FACT variant did not efficiently release from nucleosomes and was toxic in vivo Notably, intrinsic DNA-binding HMGB modules reduced the DNA accessibility and histone H2A-H2B dimer loss normally associated with nucleosome reorganization. We propose that DNA bending by HMGB domains promotes nucleosome destabilization and reorganization by exposing FACT's histone-binding sites, but DNA bending also produces DNA curvature needed to accommodate nucleosome assembly. Intrinsic DNA-bending activity therefore favors nucleosome assembly by FACT over nucleosome reorganization, but excessive activity impairs FACT release, suggesting a quality control checkpoint during nucleosome assembly.

Biochemical and Biophysical Characterisation of Higher Oligomeric Structure of Rat Nucleosome Assembly Protein 1.

Nucleosome assembly protein 1 (NAP1) is a histone chaperone that exchanges histone H2A-H2B dimer from chromatin templates. Studies with yeast NAP1 (yNAP1) have revealed itsexistence asmultiple oligomeric species in solution. Here, rat NAP1 (rNAP1), which is 98% identical to the human NAP1 (hNAP1) was used as a model to characterize the oligomeric structures of this protein in higher eukaryotes. Gel filtration chromatography and Dynamic light scattering of recombinant rNAP1 indicated that the protein exists as a complex mixture of multimeric species even at 500mM ionic strength. The solution-state complexity remains unchanged even at higher ionic strengths. Equilibrium unfolding (ΔG 14.6kcalmol- 1) shows that rNAP1, both dimeric and oligomeric, follow the two-state model of unfolding with no detectable intermediates. Homology modelling suggests that rat and yeast NAP1 share an overall similar structure with conserved domains. However, dissimilar substitutions like threonine and lysine with glycine in the β-hairpin involved in oligomerization, possibly leads to the observed differences in the oligomerization propensity of the two proteins. Molecular dynamic simulation (MDS) of the two structures also revealed that rNAP1 dimer is more stable owing to the extensive hydrogen bonding in comparison to yNAP1. Further, in vitro kinase assay showed that the phosphorylation of rNAP1 favors oligomerization with no effect on its histone binding capacity. Our results clearly suggest that there are differences in the in-solution behavior of rNAP1 compared to yNAP1 which may have in vivo functional implications for the regulation of these complexes during chromatin assembly and rearrangement.

Nucleosome acidic patch-targeting binuclear ruthenium compounds induce aberrant chromatin condensation

The ‘acidic patch’ is a highly electronegative cleft on the histone H2A–H2B dimer in the nucleosome. It is a fundamental motif for protein binding and chromatin dynamics, but the cellular impact of targeting this potentially therapeutic site with exogenous molecules remains unclear. Here, we characterize a family of binuclear ruthenium compounds that selectively target the nucleosome acidic patch, generating intra-nucleosomal H2A-H2B cross-links as well as inter-nucleosomal cross-links. In contrast to cisplatin or the progenitor RAPTA-C anticancer drugs, the binuclear agents neither arrest specific cell cycle phases nor elicit DNA damage response, but rather induce an irreversible, anomalous state of condensed chromatin that ultimately results in apoptosis. In vitro, the compounds induce misfolding of chromatin fibre and block the binding of the regulator of chromatin condensation 1 (RCC1) acidic patch-binding protein. This family of chromatin-modifying molecules has potential for applications in drug development and as tools for chromatin research.

Regulation of the Stability of the Histone H2A-H2B Dimer by H2A Tyr57 Phosphorylation.

Histone H2A and H2B form a H2A-H2B heterodimer, which is a fundamental unit of nucleosome assembly and disassembly. Several posttranslational modifications change the interface between the H2A-H2B dimer and the H3-H4 tetramer and regulate nucleosome stability. However, posttranslational modifications associated with the interface between H2A and H2B have not been discussed. In this paper, it is shown that Tyr57 phosphorylation in H2A strongly influences H2A-H2B dimerization. Tyr57-phosphorylated H2A was chemically synthesized and utilized to reconstitute the H2A-H2B dimer and nucleosome as well as canonical H2A. Thermal shift assays showed that phosphorylation destabilized the dimer and facilitated dissociation of H2A and H2B from the nucleosome structure. The proximity between H2A Tyr57 and the H2B αC helix is assumed to lead the destabilization. The DNA accessibility of the nucleosome was estimated by using micrococcal nuclease. The phosphorylated nucleosome did not change DNA accessibility compared to that of the canonical nucleosome. It is demonstrated that phosphorylation at Tyr57 changes the H2A-H2B dimer interaction but does not interfere with histone-DNA interactions. This work on the destabilization of the H2A-H2B dimer by Tyr57 phosphorylation is a promising step in elucidating control mechanisms of dynamic behavior of H2A and H2B through posttranslational modifications.

Residues in the Nucleosome Acidic Patch Regulate Histone Occupancy and Are Important for FACT Binding in Saccharomyces cerevisiae.

The essential histone chaperone FACT plays a critical role in DNA replication, repair, and transcription, primarily by binding to histone H2A-H2B dimers and regulating their assembly into nucleosomes. While FACT histone chaperone activity has been extensively studied, the exact nature of the H2A and H2B residues important for FACT binding remains controversial. In this study, we characterized the functions of residues in the histone H2A and H2B acidic patch, which is important for binding many chromatin-associated factors. We found that mutations in essential acidic patch residues cause a defect in histone occupancy in yeast, even though most of these histone mutants are expressed normally in yeast and form stable nucleosomes in vitro Instead, we show that two acidic patch residues, H2B L109 and H2A E57, are important for histone binding to FACT in vivo We systematically screened mutants in other H2A and H2B residues previously suspected to be important for FACT binding and confirmed the importance of H2B M62 using an in-vivo FACT-binding assay. Furthermore, we show that, like deletion mutants in FACT subunits, an H2A E57 and H2B M62 double mutant is lethal in yeast. In summary, we show that residues in the nucleosome acidic patch promote histone occupancy and are important for FACT binding to H2A-H2B dimers in yeast.

Single-Molecule Investigations on Histone H2A-H2B Dynamics in the Nucleosome.

Nucleosomes impose physical barriers to DNA-templated processes, playing important roles in eukaryotic gene regulation. DNA is packaged into nucleosomes by histone proteins mainly through strong electrostatic interactions that can be modulated by various post-translational histone modifications. Investigating the dynamics of histone dissociation from the nucleosome and how it is altered upon histone modifications is important for understanding eukaryotic gene regulation mechanisms. In particular, histone H2A-H2B dimer displacement in the nucleosome is one of the most important and earliest steps of histone dissociation. Two conflicting hypotheses on the requirement for dimer displacement are that nucleosomal DNA needs to be unwrapped before a dimer can displace and that a dimer can displace without DNA unwrapping. In order to test the hypotheses, we employed three-color single-molecule FRET and monitored in a time-resolved manner the early kinetics of H2A-H2B dimer dissociation triggered by high salt concentration and by histone chaperone Nap1. The results reveal that dimer displacement requires DNA unwrapping in the vast majority of the nucleosomes in the salt-induced case, while dimer displacement precedes DNA unwrapping in >60% of the nucleosomes in the Nap1-mediated case. We also found that acetylation at histone H4K16 or H3K56 affects the kinetics of Nap1-mediated dimer dissociation and facilitates the process both kinetically and thermodynamically. On the basis of these results, we suggest a mechanism by which histone chaperone facilitates H2A-H2B dimer displacement from the histone core without requiring another factor to unwrap the nucleosomal DNA.

Single-Molecule Observation Reveals Spontaneous Protein Dynamics in the Nucleosome.

Structural dynamics of a protein molecule is often critical to its function. Single-molecule methods provide efficient ways to investigate protein dynamics, although it is very challenging to achieve a millisecond or higher temporal resolution. Here we report spontaneous structural dynamics of the histone protein core in the nucleosome based on a single-molecule method that can reveal submillisecond dynamics by combining maximum likelihood estimation and fluorescence correlation spectroscopy. The nucleosome, comprising ∼147 bp DNA and an octameric histone protein core consisting of H2A, H2B, H3, and H4, is the fundamental packing unit of the eukaryotic genome. The nucleosome imposes a physical barrier that should be overcome during various DNA-templated processes. Structural fluctuation of the nucleosome in the histone core has been hypothesized to be required for nucleosome disassembly but has yet to be directly probed. Our results indicate that at 100 mM NaCl the histone H2A-H2B dimer dissociates from the histone core transiently once every 3.6 ± 0.6 ms and returns to its position within 2.0 ± 0.3 ms. We also found that the motion is facilitated upon H3K56 acetylation and inhibited upon replacing H2A with H2A.Z. These results provide the first direct examples of how a localized post-translational modification or an epigenetic variation affects the kinetic and thermodynamic stabilities of a macromolecular protein complex, which may directly contribute to its functions.

Abstract 1972: Changes in the architecture, interactions and chromatin remodeling of SWI/SNF due to loss of Snf5

Abstract The mammalian SWI/SNF complex is a family of an estimated ∼100 complexes, each with different combinations of related subunits. Efforts in sequencing many cancer genomes has revealed that mammalian SWI/SNF complexes is one of the most frequently mutated epigenetic factors found in a broad range of cancers. In order to understand more about the subunit organization of SWI/SNF and because yeast and mammalian SWI/SNF complexes share many conserved subunits and domains; we have studied the role of the Snf5 subunit in the yeast SWI/SNF to better understand the role of the mammalian homolog SNF5 that is a known tumor suppressor gene. The basis for the loss of mammalian SNF5 being a driver mutation in pediatric rhabdoid tumors is currently not well understood or how the loss of SNF5 effects the composition, recruitment and nucleosome remodeling activity of the SWI/SNF complex. We have assessed the effects on deletion of Snf5 on the compositional integrity of SWI/SNF and have evidence for Snf5 forming a sub-complex with two other highly conserved subunits of SWI/SNF that are lost from the complex when Snf5 is deleted. In addition we have mapped the inter-subunit and intra-subunit interactions of the yeast SWI/SNF complex with lysine specific homo bi-functional crosslinkers and mass spectrometry. These data confirm the formation of a tri-subunit Snf5 sub-complex and show that this module uniquely interacts with the ATPase domain of the catalytic subunit of SWI/SNF (Snf2). These observations lead to a further investigation into the regulation of the remodeling activity of SWI/SNF by the Snf5 subunit. We have found that Snf5 is required for the ATPase domain to make stable interactions with nucleosomal DNA. And the loss of these interactions in turn also causes a reduction in the remodeling efficiency of the SWI/SNF complex. We have done additional mapping of the interactions of Snf5 as part of the SWI/SNF complex and find that the highly conserved region of yeast Snf5 corresponding to the human SNF5 protein associates with the surface of the nucleosome near the histone H2A-H2B dimer. We will discuss the relevance of these findings in highlighting how an aberrant form of SWI/SNF could be created by loss of Snf5 and provide an example of how it alters the normal function and structure of SWI/SNF. Citation Format: Blaine Bartholomew, Jim Persinger, Payel Sen, Mekonnen Dechassa Lemma, Jie Luo, Jeff Ranish. Changes in the architecture, interactions and chromatin remodeling of SWI/SNF due to loss of Snf5. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1972.

Regulation of MicroRNA-Mediated Developmental Changes by the SWR1 Chromatin Remodeling Complex.

The ATP-dependent SWR1 chromatin remodeling complex (SWR1-C) exchanges the histone H2A-H2B dimer with the H2A.Z-H2B dimer, producing variant nucleosomes. Arabidopsis thaliana SWR1-C contributes to the active transcription of many genes, but also to the repression of genes that respond to environmental and developmental stimuli. Unlike other higher eukaryotic H2A.Z deposition mutants (which are embryonically lethal), Arabidopsis SWR1-C component mutants, including arp6, survive and display a pleiotropic developmental phenotype. However, the molecular mechanisms of early flowering, leaf serration, and the production of extra petals in arp6 have not been completely elucidated. We report here that SWR1-C is required for miRNA-mediated developmental control via transcriptional regulation. In the mutants of the components of SWR1-C such as arp6, sef, and pie1, miR156 and miR164 levels are reduced at the transcriptional level, which results in the accumulation of target mRNAs and associated morphological changes. Sequencing of small RNA libraries confirmed that many miRNAs including miR156 decreased in arp6, though some miRNAs increased. The arp6 mutation suppresses the accumulation of not only unprocessed primary miRNAs, but also miRNA-regulated mRNAs in miRNA processing mutants, hyl1 and serrate, which suggests that arp6 has a transcriptional effect on both miRNAs and their targets. We consistently detected that the arp6 mutant exhibits increased nucleosome occupancy at the tested MIR gene promoters, indicating that SWR1-C contributes to transcriptional activation via nucleosome dynamics. Our findings suggest that SWR1-C contributes to the fine control of plant development by generating a balance between miRNAs and target mRNAs at the transcriptional level.

Expression and purification of the full murine NPM2 and study of its interaction with protamines and histones

Mouse nucleoplasmin M.NPM2 was recombinantly expressed and the protein consisting of the complete sequence was purified and characterized. Similar to its Xenopus laevis X.NPM2 counterpart, the protein forms stable pentameric complexes and exhibits an almost undistinguishable hydrodynamic ionic strength-dependent unfolding behavior. The interaction of N.PM2 with histones and mouse P1/P2 protamines revealed that these chromosomal proteins bind preferentially to the distal part of the nucleoplasmin pentamer. Moreover, the present work highlights the critical role played by histones H2B and H4 in the association of the histone H2A-H2B dimers and histone octamer with nucleoplasmin.