DNA compaction

Fluorescence microscopy was used to investigate the conformational changes of individual T4 DNA molecules induced by different compacting agents, namely the cationic surfactants, cetyltrimethylammonium bromide (CTAB) and chloride (CTAC), iron(III), lysozyme, and protamine sulfate. A protocol for establishing size estimates is suggested to obtain reproducible results. Observations show that in the presence of lysozyme and protamine sulfate, DNA molecules exhibit a conformational change from an elongated coil structure to compact globules, usually interpreted as a first-order transition. The maximum degree of compaction that is attained when iron(III) or CTAB (CTAC) are used as compacting agents is considerably smaller, and intermediate structures (less elongated coils) are visible even for high concentrations of these agents. Dynamic light scattering experiments were carried out, for some of the systems, to assess the reliability of size estimates from fluorescence microscopy.

TriplatinNC is a highly positively charged, substitutioninert derivative of the phase II clinical anticancer drug, BBR3464. Such substitution-inert complexes form a distinct subset of polynuclear platinum complexes (PPCs) interacting with DNA and other biomolecules through noncovalent interactions. Rapid cellular entry is facilitated via interaction with cell surface glycosoaminoglycans and is a mechanism unique to PPCs. Nanoscale secondary ion mass spectrometry (nanoSIMS) showed rapid distribution within cytoplasmic and nucleolar compartments, but not the nucleus. In this article, the downstream effects of nucleolar localization are described. In human colon carcinoma cells, HCT116, the production rate of 47S rRNA precursor transcripts was dramatically reduced as an early event after drug treatment. Transcriptional inhibition of rRNA was followed by a robust G 1 arrest, and activation of apoptotic proteins caspase-8,-9, and-3 and PARP-1 in a p53-independent manner. Using cell synchronization and flow cytometry, it was determined that cells treated while in G 1 arrest immediately, but cells treated in S or G 2 successfully complete mitosis. Twenty-four hours after treatment, the majority of cells finally arrest in G 1 , but nearly one-third contained highly compacted DNA; a distinct biological feature that cannot be associated with mitosis, senescence, or apoptosis. This unique effect mirrored the efficient condensation of tRNA and DNA in cell-free systems. The combination of DNA compaction and apoptosis by TriplatinNC treatment conferred striking activity in platinum-resistant and/or p53 mutant or null cell lines. Taken together, our results support that the biological activity of TriplatinNC reflects reduced metabolic deactivation (substitution-inert compound not reactive to sulfur nucleophiles), high cellular accumulation, and novel consequences of high-affinity noncovalent DNA binding, producing a new profile and a further shift in the structure−activity paradigms for antitumor complexes.

Fluorescence microscopy was used to investigate the conformational changes of individual T4 DNA molecules induced by different compacting agents, namely the cationic surfactants, cetyltrimethylammonium bromide (CTAB) and chloride (CTAC), iron(III), lysozyme, and protamine sulfate. A protocol for establishing size estimates is suggested to obtain reproducible results. Observations show that in the presence of lysozyme and protamine sulfate, DNA molecules exhibit a conformational change from an elongated coil structure to compact globules, usually interpreted as a first-order transition. The maximum degree of compaction that is attained when iron(III) or CTAB (CTAC) are used as compacting agents is considerably smaller, and intermediate structures (less elongated coils) are visible even for high concentrations of these agents. Dynamic light scattering experiments were carried out, for some of the systems, to assess the reliability of size estimates from fluorescence microscopy.

We used poly(N-isopropylacrylamide) (PNIPAM) to control the conformation of genomic DNA by changing the temperature of a reaction solution and studied the DNA transition at the level of single DNA molecules. With this method, the conformation of long genomic DNA can be readily and reversibly switched between a very compact condensate and an unfolded macromolecule.

We recently identified the DPY19L2 gene as the main genetic cause of human globozoospermia. Non-genetically characterized cases of globozoospermia were associated with DNA alterations, suggesting that DPY19L2-dependent globozoospermia may be associated with poor DNA quality. However the origins of such defects have not yet been characterized and the consequences on the quality of embryos generated with globozoospermic sperm remain to be determined. Using the mouse model lacking Dpy19l2, we compared several key steps of nuclear compaction. We show that the kinetics of appearance and disappearance of the histone H4 acetylation waves and of transition proteins are defective. More importantly, the nuclear invasion by protamines does not occur. As a consequence, we showed that globozoospermic sperm presented with poor sperm chromatin compaction and sperm DNA integrity breakdown. We next assessed the developmental consequences of using such faulty sperm by performing ICSI. We showed in th...

TriplatinNC is a highly positively charged, substitutioninert derivative of the phase II clinical anticancer drug, BBR3464. Such substitution-inert complexes form a distinct subset of polynuclear platinum complexes (PPCs) interacting with DNA and other biomolecules through noncovalent interactions. Rapid cellular entry is facilitated via interaction with cell surface glycosoaminoglycans and is a mechanism unique to PPCs. Nanoscale secondary ion mass spectrometry (nanoSIMS) showed rapid distribution within cytoplasmic and nucleolar compartments, but not the nucleus. In this article, the downstream effects of nucleolar localization are described. In human colon carcinoma cells, HCT116, the production rate of 47S rRNA precursor transcripts was dramatically reduced as an early event after drug treatment. Transcriptional inhibition of rRNA was followed by a robust G 1 arrest, and activation of apoptotic proteins caspase-8,-9, and-3 and PARP-1 in a p53-independent manner. Using cell synchronization and flow cytometry, it was determined that cells treated while in G 1 arrest immediately, but cells treated in S or G 2 successfully complete mitosis. Twenty-four hours after treatment, the majority of cells finally arrest in G 1 , but nearly one-third contained highly compacted DNA; a distinct biological feature that cannot be associated with mitosis, senescence, or apoptosis. This unique effect mirrored the efficient condensation of tRNA and DNA in cell-free systems. The combination of DNA compaction and apoptosis by TriplatinNC treatment conferred striking activity in platinum-resistant and/or p53 mutant or null cell lines. Taken together, our results support that the biological activity of TriplatinNC reflects reduced metabolic deactivation (substitution-inert compound not reactive to sulfur nucleophiles), high cellular accumulation, and novel consequences of high-affinity noncovalent DNA binding, producing a new profile and a further shift in the structure−activity paradigms for antitumor complexes.

The mammalian cluster lies between two topologically associating domains (TADs) matching distinct enhancer-rich regulatory landscapes. During limb development, the telomeric TAD controls the early transcription of genes in forearm cells, whereas the centromeric TAD subsequently regulates more posterior genes in digit cells. Therefore, the TAD boundary prevents the terminal gene from responding to forearm enhancers, thereby allowing proper limb patterning. To assess the nature and function of this CTCF-rich DNA region in embryos we compared chromatin interaction profiles between proximal and distal limb bud cells isolated from mutant stocks where various parts of this boundary region were removed. The resulting progressive release in boundary effect triggered inter-TAD contacts, favored by the activity of the newly accessed enhancers. However, the boundary was highly resilient, and only a 400-kb deletion, including the whole-gene cluster, was eventually able to merge the neighboring ...

Chromosome conformation capture (3C)-based techniques have revolutionized the field of nuclear organization, partly replacing DNA FISH as the method of choice for studying three-dimensional chromosome architecture. Although DNA FISH is commonly used for confirming 3C-based findings, the two techniques are conceptually and technically different and comparing their results is not trivial. Here, we discuss both 3C-based techniques and DNA FISH approaches to highlight their similarities and differences. We then describe the technical biases that affect each approach, and review the available reports that address their compatibility. Finally, we propose an experimental scheme for comparison of 3C and DNA FISH results.

Chromosome conformation capture (3C)-based techniques such as 5C and Hi-C revealed that the folding of mammalian chromosomes is highly hierarchical. A fundamental structural unit in the hierarchy is represented by topologically associating domain (TADs), sub-megabase regions of the genome within which the chromatin fibre preferentially interacts. 3C-based methods provide the mean contact probabilities between chromosomal loci, averaged over a large number of cells, and do not give immediate access to the single-cell conformations of the chromatin fibre. However, coarse-grained polymer models based on 5C data can be used to extract the single-cell conformations of single TADs. Here we extend this approach to analyse around 2500 TADs in murine embryonic stem cells based on high-resolution Hi-C data. This allowed to predict the cell-to-cell variability in single contacts within TADs genome-wide, and correlations between them. Based on these results, we predict that TADs are more similar to ideal chains than to globules in terms of their physical size and three-dimensional shape distribution. Furthermore, we show that their physical size and the degree of structural anisotropy of single TADs are correlated with the level of transcriptional activity of the genes that it harbours. Finally, we show that a large number of multiplets of genomic loci co-localise more often than expected by random, and these loci are particularly enriched in promoters, enhancers and CTCFbound sites. These results provide the first genome-wide structural reconstruction of TADs using polymeric models obeying the laws of thermodynamics, and reveal important universal trends in the correlation between chromosome structure and transcription.

In this work, the influence of the cyclodextrin, CD, nature on the decompaction of positively charged compacted dimeric surfactant-DNA complexes was investigated. First, the condensation of calf thymus DNA by addition of three cationic dimeric surfactants with different spacer groups was studied by fluorescence, zeta potential, circular dichroism and atomic force microscopy measurements. Electromotive force experiments provided quantitative information about the influence of the spacer group on the DNA surfactant compaction efficiency. Cytotoxicity was evaluated to determine the biocompatibility of the cationic lipids. Subsequently, the decompaction of the surfactant-DNA complexes was achieved by adding α-, β-, and γ-cyclodextrin, the experimental observations being analogous for the three surfactants investigated. αand β-cyclodextrin were found to behave similarly. These CDs completely hinder the interactions between the surfactant and the nucleic acid and provoke the DNA morphological change from a globular to an elongated form. A concentration of γ-CD higher than those of αor β-CD is necessary in order to decompact the nucleic acid. Besides, zeta potential measurements show that in the presence of γ-CD, surfactant-DNA interactions are only partially hindered, some of the surfactant molecules remaining bound to the DNA.

We present experimental results on the interaction of DNA macromolecules with cationic lipid membranes with different properties, including freestanding membranes in the fluid and gel state, and supported lipid membranes in the fluid state and under conditions of fluid-gel phase coexistence. We observe diverse conformational dynamics of membrane-bound DNA molecules controlled by the local properties of the lipid bilayer. In case of fluid-state freestanding lipid membranes, the behaviour of DNA on the membrane is controlled by the membrane charge density: whereas DNA bound to weakly charged membranes predominantly behaves as a 2D random coil, an increase in the membrane charge density leads to membrane-driven irreversible DNA collapse and formation of subresolution-sized DNA globules. On the other hand, electrostatic binding of DNA macromolecules to gel-state freestanding membranes leads to completely arrested diffusion and conformational dynamics of membrane-adsorbed DNA. A drastically different picture is observed in case of DNA interaction with supported cationic lipid bilayers: When the supported bilayer is in the fluid state, membrane-bound DNA molecules undergo 2D translational Brownian motion and conformational fluctuations, irrespectively of the charge density of the supported bilayer. At the same time, when the supported cationic membrane shows fluid-gel phase coexistence, membrane-bound DNA molecules are strongly attracted to micrometre-sized gel-phase domains enriched with the cationic lipid, which results in 2D compaction of the membrane-bound macromolecules. This DNA compaction, however, is fully reversible, and disappears as soon as the membrane is heated above the fluid-gel coexistence. We also discuss possible biological implications of our experimental findings.

The genome is organized in three-dimensional units called topologically associating domains (TADs), through a process dependent on the cooperative action of cohesin and the DNA-binding factor CTCF. Genomic rearrangements of TADs have been shown to cause gene misexpression and disease, but genome-wide depletion of CTCF has no drastic effects on transcription. Here, we investigate TAD function in vivo in mouse limb buds at the Sox9-Kcnj2 locus. We show that the removal of all major CTCF sites at the boundary and within the TAD resulted in a fusion of neighboring TADs, without major effects on gene expression. Gene misexpression and disease phenotypes, however, were achieved by redirecting regulatory activity through inversions and/or the repositioning of boundaries. Thus, TAD structures provide robustness and precision but are not essential for developmental gene regulation. Aberrant disease-related gene activation is not induced by a mere loss of insulation but requires CTCFdependent redirection of enhancer-promoter contacts.

In the present work, we show a new approach for decompaction of DNA-cationic surfactant complexes, e.g., lipoplexes, by using-cyclodextrin (-CD). The DNA decompaction was achieved by dissolving the surfactant aggregates in the complex by making use of the high affinity between the-CD and the free surfactant in solution. The results from fluorescence microscopy and adiabatic compressibility measurements indicate that coils and globules do not coexist. The reported procedure using-CD is an efficient way to decompact DNA surfactant complexes because the association constant of surfactants with-CD is large. The surfactant's interaction with-CD is specific and the nonspecific interaction between-CD and biological interfaces is small.

Electrophoretic properties of complexes between DNA and the cationic surfactant cetyltrimethylammonium bromide We use agarose gel electrophoresis to characterize how the monovalent catioinic surfactant cetyltrimethylammonium bromide (CTAB) compacts double-stranded DNA, which is detected as a reduction in electrophoretic DNA velocity. The velocity reaches a plateau at a ratio R = 1.8 of CTAB to DNA-phosphate charges, i.e., above the neutralization point, and the complexes retain a net negative charge at least up to R = 200. Condensation experiments on a mixture of two DNA sizes show that the complexes formed contain only one condensed DNA molecule each. These CTAB-DNA globules were further characterized by time-resolved measurements of their velocity inside the gel, which showed that CTAB does not dissociate during the migration but possibly upon entry into the gel. Using the Ogston-model for electrophoresis of spherical particles, the measured in-gel velocity of the globule is quantitatively consistent with CTAB having two opposite effects, reduction of both the electrophoretic charge and DNA coil size. In the case of CTAB the two effects nearly cancel, which can explain why opposite velocity shifts (globule faster than uncomplexed DNA) have been observed with some catioinic condensation agents. Dissociation of the complexes by addition of anionic surfactants was also studied. The DNA release from the globule was complete at a mixing ratio between anionic and cationic surfactants equal to 1, in agreement with equilibrium studies. Circular DNA retained its supercoiling, and this demonstrates a lack of DNA nicking in the compaction-release cycle which is important in DNA transfection and purification applications.

A pentavalent branched-chain polyamine, N -bis(aminopropyl)spermidine 3(3)(3)4, is a unique polycation found in the hyperthermophilic archaeon Thermococcus kodakarensis, which grows at temperatures between 60 and 100 °C. We studied the effects of this branched-chain polyamine on DNA structure at different temperatures up to 80 °C. Atomic force microscopic observation revealed that 3(3)(3)4 induces a mesh-like structure on a large DNA (166 kbp) at 24 °C. With an increase in temperature, DNA molecules tend to unwind, and multiple nano-loops with a diameter of 10-50 nm are generated along the DNA strand at 80 °C. These results were compared to those obtained with linear-chain polyamines, homocaldopentamine 3334 and spermidine, the former of which is a structural isomer of 3(3)(3)4. These specific effects are expected to neatly concern with its role on high-temperature preference in hyperthermophiles.

The higher-order structure of compacted single giant DNA induced by complexation with polypeptide (poly-Arg) in NaCI solution was investigated using fluorescence microscopy. As the poly-Arg concentration increased, the mean size of extended DNA chains gradually decreased. In the presence of excess poly-Arg, individual DNA chains collapsed into compact globules, and the degree of collapse of the DNA chains depended not only on the concentration of poly-Arg, but also on the time course of the addition of poly-Arg and NaCI, indicating that the structure of the collapsed DNA is not determined simply according to the minimum free energy. We discuss theoretically the presence of multiple-stationary states based on a consideration of simple kinetics in the process of binding. Depending on the past history, the number of poly-Arg and Na+ that bind to each DNA changes markedly. This interesting characteristic of long DNA is discussed in relation to the possible mechanism of self-regulation of gene expression in living cells.

Deciphering the rules of genome folding in the cell nucleus is essential to understand its functions. Recent chromosome conformation capture (Hi-C) studies have revealed that the genome is partitioned into topologically associating domains (TADs), which demarcate functional epigenetic domains defined by combinations of specific chromatin marks. However, whether TADs are true physical units in each cell nucleus or whether they reflect statistical frequencies of measured interactions within cell populations is unclear. Using a combination of Hi-C, three-dimensional (3D) fluorescent in situ hybridization, super-resolution microscopy, and polymer modeling, we provide an integrative view of chromatin folding in Drosophila. We observed that repressed TADs form a succession of discrete nanocompartments, interspersed by less condensed active regions. Single-cell analysis revealed a consistent TAD-based physical compartmentalization of the chromatin fiber, with some degree of heterogeneity in intra-TAD conformations and in cis and trans inter-TAD contact events. These results indicate that TADs are fundamental 3D genome units that engage in dynamic higher-order inter-TAD connections. This domain-based architecture is likely to play a major role in regulatory transactions during DNA-dependent processes.

A statistical analysis of AFM images of DNA–surfactant complexes passed into chloroform from the aqueous phase was performed. A universal mathematical model that made it possible to reconstruct the true geometry of objects from their AFM image was developed. This model was used to determine the number of DNA molecules in individual complexes. The histograms of the distribution of complexes over the number of molecules in the complex were constructed, which showed that 80% of the DNA–surfactant complexes contained from 2 to 15 DNA molecules.

The L phase of the ternary CTAB/benzyl alcohol/water system that extends from the water corner to the alcohol corner was studied by the sound velocity method which indicated variations in velocity when the aggregate structure changed from normal micelles in water to reverse micelles in the alcohol corner of the phase diagram. From these results it was concluded that the transition from normal to reverse micelles takes place while passing through two different bicontinuous regions, one of them with alcohol micro domains dispersed in water and the other one as a bicontinuous reverse region with the predominance of the water domains dispersed in alcohol.

Chromatin condensation plays an important role in the regulation of gene expression. Recently, it was shown that the transcriptional activation of Hoxd genes during vertebrate digit development involves modifications in 3D interactions within and around the HoxD gene cluster. This reorganization follows a global transition from one set of regulatory contacts to another, between two topologically associating domains (TADs) located on either side of
the HoxD locus. Here, we use 3D DNA FISH to assess the spatial
organization of chromatin at and around the HoxD gene cluster
and report that although the two TADs are tightly associated, they
appear as spatially distinct units. We measured the relative position of genes within the cluster and found that they segregate over long distances, suggesting that a physical elongation of the
HoxD cluster can occur. We analyzed this possibility by super-resolution imaging (STORM) and found that tissues with distinct transcriptional activity exhibit differing degrees of elongation. We also observed that the morphological change of the HoxD cluster in developing digits is associated with its position at the boundary between the two TADs. Such variations in the fine-scale architecture of the gene cluster suggest causal links among its spatial configuration, transcriptional activation, and the flanking chromatin context.

The compaction of high-molecular weight DNA T4 in a water-alcohol medium has been studied by AFM. The AFM images of compact globules formed by DNA molecules in water-alcohol media have been obtained . It has been found that at 40-50% alcohol concentrations, the DNA molecules form partially compacted formations in which particular coils of the macromolecule are twisted to form toroidal structures. Using the procedure of the recovery of the true geometric parameters of the object by the AFM profile. we have showed that the globule contains one DNA molecule. The model of DNA packing during the compaction has been proposed.

In the present work, we show a new approach for decompaction of DNA-cationic surfactant complexes, e.g., lipoplexes, by using -cyclodextrin ( -CD). The DNA decompaction was achieved by dissolving the surfactant aggregates in the complex by making use of the high affinity between the -CD and the free surfactant in solution. The results from fluorescence microscopy and adiabatic compressibility measurements indicate that coils and globules do not coexist. The reported procedure using -CD is an efficient way to decompact DNA surfactant complexes because the association constant of surfactants with -CD is large. The surfactant's interaction with -CD is specific and the nonspecific interaction between -CD and biological interfaces is small.

In the present work, we show a new approach for decompaction of DNA-cationic surfactant complexes, e.g., lipoplexes, by using -cyclodextrin ( -CD). The DNA decompaction was achieved by dissolving the surfactant aggregates in the complex by making use of the high affinity between the -CD and the free surfactant in solution. The results from fluorescence microscopy and adiabatic compressibility measurements indicate that coils and globules do not coexist. The reported procedure using -CD is an efficient way to decompact DNA surfactant complexes because the association constant of surfactants with -CD is large. The surfactant's interaction with -CD is specific and the nonspecific interaction between -CD and biological interfaces is small.