DNA-mediated Processes Research Articles

Contribution of variant subunits and associated factors to genome-wide distribution and dynamics of cohesin

BackgroundThe cohesin complex organizes the genome-forming dynamic chromatin loops that impact on all DNA-mediated processes. There are two different cohesin complexes in vertebrate somatic cells, carrying the STAG1 or STAG2 subunit, and two versions of the regulatory subunit PDS5, PDS5A and PDS5B. Mice deficient for any of the variant subunits are embryonic lethal, which indicates that they are not functionally redundant. However, their specific behavior at the molecular level is not fully understood.ResultsThe genome-wide distribution of cohesin provides important information with functional consequences. Here, we have characterized the distribution of cohesin subunits and regulators in mouse embryo fibroblasts (MEFs) either wild type or deficient for cohesin subunits and regulators by chromatin immunoprecipitation and deep sequencing. We identify non-CTCF cohesin-binding sites in addition to the commonly detected CTCF cohesin sites and show that cohesin-STAG2 is the preferred variant at these positions. Moreover, this complex has a more dynamic association with chromatin as judged by fluorescence recovery after photobleaching (FRAP), associates preferentially with WAPL and is more easily extracted from chromatin with salt than cohesin-STAG1. We observe that both PDS5A and PDS5B are exclusively located at cohesin-CTCF positions and that ablation of a single paralog has no noticeable consequences for cohesin distribution while double knocked out cells show decreased accumulation of cohesin at all its binding sites. With the exception of a fraction of cohesin positions in which we find binding of all regulators, including CTCF and WAPL, the presence of NIPBL and PDS5 is mutually exclusive, consistent with our immunoprecipitation analyses in mammalian cell extracts and previous results in yeast.ConclusionOur findings support the idea that non-CTCF cohesin-binding sites represent sites of cohesin loading or pausing and are preferentially occupied by the more dynamic cohesin-STAG2. PDS5 proteins redundantly contribute to arrest cohesin at CTCF sites, possibly by preventing binding of NIPBL, but are not essential for this arrest. These results add important insights towards understanding how cohesin regulates genome folding and the specific contributions of the different variants that coexist in the cell.

MESA: automated assessment of synthetic DNA fragments and simulation of DNA synthesis, storage, sequencing and PCR errors.

SummaryThe development of de novo DNA synthesis, polymerase chain reaction (PCR), DNA sequencing and molecular cloning gave researchers unprecedented control over DNA and DNA-mediated processes. To reduce the error probabilities of these techniques, DNA composition has to adhere to method-dependent restrictions. To comply with such restrictions, a synthetic DNA fragment is often adjusted manually or by using custom-made scripts. In this article, we present MESA (Mosla Error Simulator), a web application for the assessment of DNA fragments based on limitations of DNA synthesis, amplification, cloning, sequencing methods and biological restrictions of host organisms. Furthermore, MESA can be used to simulate errors during synthesis, PCR, storage and sequencing processes.Availability and implementationMESA is available at mesa.mosla.de, with the source code available at github.com/umr-ds/mesa_dna_sim.Contact dominik.heider@uni-marburg.de Supplementary information Supplementary data are available at Bioinformatics online.

Transient Kinetic Analysis of SWR1C-Catalyzed H2A.Z Deposition Unravels the Impact of Nucleosome Dynamics and the Asymmetry of Histone Exchange

SUMMARYThe SWR1C chromatin remodeling enzyme catalyzes ATP-dependent replacement of nucleosomal H2A with the H2A.Z variant, regulating key DNA-mediated processes such as transcription and DNA repair. Here, we investigate the transient kinetic mechanism of the histone exchange reaction, employing ensemble FRET, fluorescence correlation spectroscopy (FCS), and the steady-state kinetics of ATP hydrolysis. Our studies indicate that SWR1C modulates nucleosome dynamics on both the millisecond and microsecond timescales, poising the nucleosome for the dimer exchange reaction. The transient kinetic analysis of the remodeling reaction performed under single turnover conditions unraveled a striking asymmetry in the ATP-dependent replacement of nucleosomal dimers, promoted by localized DNA unwrapping. Taken together, our transient kinetic studies identify intermediates and provide crucial insights into the SWR1C-catalyzed dimer exchange reaction and shed light on how the mechanics of H2A.Z deposition might contribute to transcriptional regulation in vivo.

Defining the Structure of a Protein-Spherical Nucleic Acid Conjugate and Its Counterionic Cloud.

Protein–spherical nucleic acid conjugates (Pro-SNAs) are an emerging class of bioconjugates that have properties defined by their protein cores and dense shell of oligonucleotides. They have been used as building blocks in DNA-driven crystal engineering strategies and show promise as agents that can cross cell membranes and affect both protein and DNA-mediated processes inside cells. However, ionic environments surrounding proteins can influence their activity and conformational stability, and functionalizing proteins with DNA substantively changes the surrounding ionic environment in a nonuniform manner. Techniques typically used to determine protein structure fail to capture such irregular ionic distributions. Here, we determine the counterion radial distribution profile surrounding Pro-SNAs dispersed in RbCl with 1 nm resolution through in situ anomalous small-angle X-ray scattering (ASAXS) and classical density functional theory (DFT). SAXS analysis also reveals the radial extension of the DNA and the linker used to covalently attach the DNA to the protein surface. At the experimental salt concentration of 50 mM RbCl, Rb+ cations compensate ∼90% of the negative charge due to the DNA and linker. Above 75 mM, DFT calculations predict overcompensation of the DNA charge by Rb+. This study suggests a method for exploring Pro-SNA structure and function in different environments through predictions of ionic cloud densities as a function of salt concentration, DNA grafting density, and length. Overall, our study demonstrates that solution X-ray scattering combined with DFT can discern counterionic distribution and submolecular features of highly charged, complex nanoparticle constructs such as Pro-SNAs and related nucleic acid conjugate materials.

Role of Protein Phosphorylation in the Regulation of Cell Cycle and DNA-Related Processes in Bacteria.

In all living organisms, the phosphorylation of proteins modulates various aspects of their functionalities. In eukaryotes, protein phosphorylation plays a key role in cell signaling, gene expression, and differentiation. Protein phosphorylation is also involved in the global control of DNA replication during the cell cycle, as well as in the mechanisms that cope with stress-induced replication blocks. Similar to eukaryotes, bacteria use Hanks-type kinases and phosphatases for signal transduction, and protein phosphorylation is involved in numerous cellular processes. However, it remains unclear whether protein phosphorylation in bacteria can also regulate the activity of proteins involved in DNA-mediated processes such as DNA replication or repair. Accumulating evidence supported by functional and biochemical studies suggests that phospho-regulatory mechanisms also take place during the bacterial cell cycle. Recent phosphoproteomics and interactomics studies identified numerous phosphoproteins involved in various aspect of DNA metabolism strongly supporting the existence of such level of regulation in bacteria. Similar to eukaryotes, bacterial scaffolding-like proteins emerged as platforms for kinase activation and signaling. This review reports the current knowledge on the phosphorylation of proteins involved in the maintenance of genome integrity and the regulation of cell cycle in bacteria that reveals surprising similarities to eukaryotes.

Quantitative nanoscale imaging of orientational order in biological filaments by polarized superresolution microscopy

Essential cellular functions as diverse as genome maintenance and tissue morphogenesis rely on the dynamic organization of filamentous assemblies. For example, the precise structural organization of DNA filaments has profound consequences on all DNA-mediated processes including gene expression, whereas control over the precise spatial arrangement of cytoskeletal protein filaments is key for mechanical force generation driving animal tissue morphogenesis. Polarized fluorescence is currently used to extract structural organization of fluorescently labeled biological filaments by determining the orientation of fluorescent labels, however with a strong drawback: polarized fluorescence imaging is indeed spatially limited by optical diffraction, and is thus unable to discriminate between the intrinsic orientational mobility of the fluorophore labels and the real structural disorder of the labeled biomolecules. Here, we demonstrate that quantitative single-molecule polarized detection in biological filament assemblies allows not only to correct for the rotational flexibility of the label but also to image orientational order of filaments at the nanoscale using superresolution capabilities. The method is based on polarized direct stochastic optical reconstruction microscopy, using dedicated optical scheme and image analysis to determine both molecular localization and orientation with high precision. We apply this method to double-stranded DNA in vitro and microtubules and actin stress fibers in whole cells.

H2A.Z: a molecular rheostat for transcriptional control.

The replacement of nucleosomal H2A with the histone variant H2A.Z is critical for regulating DNA-mediated processes across eukaryotes and for early development of multicellular organisms. How this variant performs these seemingly diverse roles has remained largely enigmatic. Here, we discuss recent mechanistic insights that have begun to reveal how H2A.Z functions as a molecular rheostat for gene control. We focus on specific examples in metazoans as a model for understanding how H2A.Z integrates information from histone post-translational modifications, other histone variants, and transcription factors (TFs) to regulate proper induction of gene expression programs in response to cellular cues. Finally, we propose a general model of how H2A.Z incorporation regulates chromatin states in diverse processes.

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.

A novel route to product specificity in the Suv4-20 family of histone H4K20 methyltransferases

The delivery of site-specific post-translational modifications to histones generates an epigenetic regulatory network that directs fundamental DNA-mediated processes and governs key stages in development. Methylation of histone H4 lysine-20 has been implicated in DNA repair, transcriptional silencing, genomic stability and regulation of replication. We present the structure of the histone H4K20 methyltransferase Suv4-20h2 in complex with its histone H4 peptide substrate and S-adenosyl methionine cofactor. Analysis of the structure reveals that the Suv4-20h2 active site diverges from the canonical SET domain configuration and generates a high degree of both substrate and product specificity. Together with supporting biochemical data comparing Suv4-20h1 and Suv4-20h2, we demonstrate that the Suv4-20 family enzymes take a previously mono-methylated H4K20 substrate and generate an exclusively di-methylated product. We therefore predict that other enzymes are responsible for the tri-methylation of histone H4K20 that marks silenced heterochromatin.

Nucleosomal DNA binding drives the recognition of H3K36-methylated nucleosomes by the PSIP1-PWWP domain

BackgroundRecognition of histone modifications by specialized protein domains is a key step in the regulation of DNA-mediated processes like gene transcription. The structural basis of these interactions is usually studied using histone peptide models, neglecting the nucleosomal context. Here, we provide the structural and thermodynamic basis for the recognition of H3K36-methylated (H3K36me) nucleosomes by the PSIP1-PWWP domain, based on extensive mutational analysis, advanced nuclear magnetic resonance (NMR), and computational approaches.ResultsThe PSIP1-PWWP domain binds H3K36me3 peptide and DNA with low affinity, through distinct, adjacent binding surfaces. PWWP binding to H3K36me nucleosomes is enhanced approximately 10,000-fold compared to a methylated peptide. Based on mutational analyses and NMR data, we derive a structure of the complex showing that the PWWP domain is bound to H3K36me nucleosomes through simultaneous interactions with both methylated histone tail and nucleosomal DNA.ConclusionConcerted binding to the methylated histone tail and nucleosomal DNA underlies the high- affinity, specific recognition of H3K36me nucleosomes by the PSIP1-PWWP domain. We propose that this bipartite binding mechanism is a distinctive and general property in the recognition of histone modifications close to the nucleosome core.

Comparative genome analysis suggests characteristics of yeast inverted repeats that are important for transcriptional activity

Inverted repeats are sequences of DNA that, when read in the 5' to 3' direction, have the same sequence on both strands (palindromic portion), with the exception of a small number of nucleotides in the exact center (nonpalindromic spacer). They have been implicated in various DNA-mediated processes including replication, transcription, and genomic instability. At least some of these sequences are capable of forming an alternative DNA structure, called a cruciform, that may be important for mediating these functions. We generated a list of inverted repeats in the Saccharomyces cerevisiae genome and determined which of them are conserved in three related yeasts. We have identified characterisitics of inverted repeats that make them more likely to be conserved than the surrounding DNA and characteristics, such as position and base composition, that make the genes they are associated with likely to be more actively transcribed. This is an important step in determining the functions of this group of genomic elements.

The Histone Modifications Governing TFF1 Transcription Mediated by Estrogen Receptor

Transcription regulation by histone modifications is a major contributing factor to the structural and functional diversity in biology. These modifications are encrypted as histone codes or histone languages and function to establish and maintain heritable epigenetic codes that define the identity and the fate of the cell. Despite recent advances revealing numerous histone modifications associated with transcription regulation, how such modifications dictate the process of transcription is not fully understood. Here we describe spatial and temporal analyses of the histone modifications that are introduced during estrogen receptor α (ERα)-activated transcription. We demonstrated that aborting RNA polymerase II caused a disruption of the histone modifications that are associated with transcription elongation but had a minimal effect on modifications deposited during transcription initiation. We also found that the histone H3S10 phosphorylation mark is catalyzed by mitogen- and stress-activated protein kinase 1 (MSK1) and is recognized by a 14-3-3ζ/14-3-3ε heterodimer through its interaction with H3K4 trimethyltransferase SMYD3 and the p52 subunit of TFIIH. We showed that H3S10 phosphorylation is a prerequisite for H3K4 trimethylation. In addition, we demonstrated that SET8/PR-Set7/KMT5A is required for ERα-regulated transcription and its catalyzed H4K20 monomethylation is implicated in both transcription initiation and elongation. Our experiments provide a relatively comprehensive analysis of histone modifications associated with ERα-regulated transcription and define the biological meaning of several key components of the histone code that governs ERα-regulated transcription.

In nucleo enzymatic assays for the identification and characterization of histone modifying activities

The impact of histone phosphorylation, acetylation, and methylation onto transcription and various other cellular DNA-mediated processes is now well established. Numerous histone modification marks, specific sites carrying particular post-translational modifications, have been described and analyzed in detail. Whereas, many methods for the study of histone post-translational modifications involve sophisticated equipment and techniques, we describe the revival of a very simple procedure for the analysis of histone modifications and histone modifying enzymes, which is derived from experiments first carried out several decades ago. This method is based on the isolation of cell nuclei containing intact chromatin structures that are then incubated with defined enzymatic substrates of histone modifying enzymes. We provide quick protocols for the isolation of nuclei from yeast and mammalian cells and give basic procedures for the phosphorylation, acetylation, and methylation of histones (and other proteins) using these subcellular sources that can be carried out in any laboratory. Simple methods for the analysis of histone modifications using these assays are discussed.

A Conserved Swi2/Snf2 ATPase Motif Couples ATP Hydrolysis to Chromatin Remodeling

Yeast (Saccharomyces cerevisiae) SWI/SNF is a prototype for a large family of ATP-dependent chromatin-remodeling enzymes that facilitate numerous DNA-mediated processes. Swi2/Snf2 is the catalytic subunit of SWI/SNF, and it is the founding member of a novel subfamily of the SF2 superfamily of DNA helicase/ATPases. Here we present a functional analysis of the diagnostic set of helicase/ATPase sequence motifs found within all Swi2p/Snf2p family members. Whereas many of these motifs play key roles in ATP binding and/or hydrolysis, we identify residues within conserved motif V that are specifically required to couple ATP hydrolysis to chromatin-remodeling activity. Interestingly, motif V of the human Swi2p/Snf2p homolog, Brg1p, has been shown to be a possible hot spot for mutational alterations associated with cancers.

The SANT domain: a unique histone-tail-binding module?

Chromatin-remodelling complexes have an important role in all DNA-mediated processes and, although much is known about how these enzymes regulate chromosomal DNA accessibility, how they interact with their histone substrates has remained unclear. However, recent studies have indicated that the SANT domain has a central role in chromatin remodelling by functioning as a unique histone-interaction module that couples histone binding to enzyme catalysis.

Structural Basis for Topoisomerase I Inhibition by Nucleoside Analogs

Nucleoside analogs such as 1-β-D-arabinofuranosyl cytidine (AraC) and 2′, 2′-difluoro deoxycytidine (dFdC) are important components of the anticancer chemotherapeutic arsenal and are among the most effective anticancer drugs currently available. Although both AraCTP and dFdCTP impede DNA replication through pausing of DNA polymerases, both nucleoside analogs are ultimately incorporated into replicated DNA and interfere in DNA-mediated processes. Our laboratories are investigating the structural basis for the poisoning of topoisomerase I (top1) due to antipyrimidine incorporation into duplex DNA. We recently reported that both AraC and dFdC induce formation of top1 cleavage complexes, and poisoning of top1 contributes to the anticancer activities of both these drugs. Recent NMR and thermodynamic studies from our laboratories provide insight into the mechanism by which AraC and dFdC poison top 1. NMR studies from our laboratories have revealed that the arabinosyl sugar of AraC adopted a C2′-endo conformation. Although this is a B-type sugar pucker characteristic of duplex DNA, the conformation is rigid, and this lack of flexibility probably contributes to inhibition of the religation step of the top 1 reaction. In contrast to AraC, NMR studies revealed dFdC adopted a C3′ endo sugar pucker characteristic of RNA, rather than DNA duplexes. dFdC substitution enhanced formation of top1 cleavage complexes, but did not inhibit religation. The enhancement of top1 cleavage complexes most likely results from a combination of conformational and electrostatic effects. The structural effects of dFdC and AraC are being further investigated in duplex DNA with well-defined top1 cleavage sites to analyze more specifically how these structural perturbations lead to enzyme poisoning.

In vivo assays to study histone ubiquitylation

The importance of histone acetylation, phosphorylation, and methylation in transcription and other DNA-mediated processes is now well established. Histones are also ubiquitylated, but in contrast to the majority of ubiquitylated proteins, ubiquitylated histones are not generally targeted for degradation and may play roles similar to those of other histone modifications. Antibodies against acetylated histones have provided unique insights into the regulation, distribution, and cellular roles of these modified histones. In this report, we describe methods to identify ubiquitylated histones in budding yeast and HeLa cells. We provide protocols to detect ubiquitylated histones that are based on a combination of in vivo genetic and immunological assays. These methods should provide relatively simple and useful tools to study the global regulation of this important but poorly understood histone modification.

NMR structure of a gemcitabine-substituted model Okazaki fragment.

Gemcitabine (2'-deoxy-2',2'-difluorodeoxycytidine; dFdC) is a potent anticancer drug that exerts cytotoxic activity, in part, through incorporation of the nucleoside triphosphate dFdCTP into DNA and perturbations to DNA-mediated processes. The structure of a model Okazaki fragment containing a single dFdC substitution, [GEM], was determined using NMR spectroscopy and restrained molecular dynamics to understand structural distortions that may be induced in replicating DNA resulting from dFdC substitution. The electrostatic surface of [GEM] was also computed to determine how the geminal difluoro group of dFdC perturbs DNA electrostatics. The stability of [GEM] was investigated using temperature-dependent UV spectroscopy. dFdC adopted a C3'-endo conformation in [GEM] and decreased the melting temperature of the duplex by 4.3 degrees C. dFdC substitution did not decrease helical stacking among adjacent purines in the DNA duplex region. dFdC substitution substantially altered the electrostatic properties of the model Okazaki fragment, with increased electron density in the vicinity of the geminal difluoro group. The results are consistent with dFdC substitution altering the structural, electrostatic, and thermodynamic properties of DNA and interfering in DNA-mediated processes. Interference in DNA-mediated processes due to dFdC substitution likely contributes to the anticancer activity of dFdC.

ATP-dependent chromatin remodeling: going mobile

Members of the ATP-dependent class of chromatin remodeling enzymes are found in all eukaryotes where they play key roles in many DNA-mediated processes. Each of these enzymes are multi-subunit assembles that hydrolyze ∼1000 ATP/min. The energy of ATP hydrolysis is used to disrupt the chromatin structure which can be scored by enhanced factor binding, disruption of the DNase I cleavage pattern of mononucleosomes, formation of dinucleosomes, movements of histone octamers in cis and in trans, and by generation of nuclease hypersensitive sites. Here the biochemical properties of these enzymes are reviewed and the manner in which ATP-driven nucleosome movements might account for many of these diverse activities is discussed.