Effects of fast neutrons on chromatin: dependence on chromatin structure

The effects of fast neutrons (10-100 Gy) on chromatin extracted from normal (liver of Wistar rats) and tumor (Walker carcinosarcoma maintained on Wistar rats) tissues were compared. The spectroscopic assays used were (i) chromatin intrinsic fluorescence, (ii) time-resolved fluorescence of chromatin - proflavine complexes, and (iii) fluorescence resonance energy transfer (FRET) between dansyl chloride and acridine orange coupled to chromatin. For both normal and tumor chromatin, the intensity of intrinsic fluorescence specific for acidic and basic proteins decreased with increasing dose. The relative contributions of the excited-state lifetime of proflavine bound to chromatin were reduced upon fast-neutron irradiation, indicating a decrease in the proportion of chromatin DNA available for ligand binding. The Forster energy transfer efficiencies were also modified by irradiation. These effects were larger for chromatin from tumor tissue. In the range 0-100 Gy, fast neutrons induced alterations in DNA and acidic and basic proteins, as well as in global chromatin structure. The radiosensitivity of chromatin extracted from tumor tissue seems to be higher than that of chromatin extracted from normal tissue, probably because of its higher euchromatin (loose)--heterochromatin (compact) ratio.     

Studies on chromatin. II. Isolation and characterization of chromatin subunits.

Earlier findings /1-10/ bearing on a subunit organization of chromatin were confirmed and in some points detailed. Besides this, a large-scale isolation of chromatin subunits; their protein composition, electron microscopic appearance and CsCl banding pattern are described. Although the purified chromatin subunit contains all five histones, the relative content of histone H1 i in it is two times lower than that in the original chromatin. tit is shown that a mild digestion of chromatin with staphylococcal nuclease produced not only separate chromatin subunits and their "oligomers' but also deoxyribonucleoprotein particles which sediment more slowly than subunits. It appears that these particles and subunits are produced from different initial structures in the chromatin. Finally, a crystallization of the purified chromatin subunit as a cetyltrimethyl ammonium salt is described.     

ATP依赖的染色质改构复合物及其作用机制

染色质高度紧密的折叠阻止了转录因子和辅因子与DNA的结合, 因而通过染色质重塑以解除这样的抑制环境, 对于转录活动的正常进行是至关重要的。目前认为, 染色质重塑至少是通过两种机制来完成的, 一种是通过ATP依赖的染色质改构复合物, 另一种是通过对组蛋白尾部进行共价修饰的组蛋白修饰酶复合物。文章结合近年来的研究进展, 对前者进行染色质重塑的机制及两者在基因转录调控过程中如何相互协作等进行了论述。     

Studies on chromatin

Chromatin lacking histone H1 was found by electron microscopy to contain 'beaded' deoxyribonucleoprotein fibers. Adjacent beads are connected with each other by threads having a DNA-like appearance. At least some of threads are shown to be free DNA stretches. Average length and the content of free DNA stretches in histone H1-depleted chromatin depends on the ionic conditions of the medium. The appearance of individual beads is similar to that of chromatin subunits or v-bodies [1] in the original chromatin. Thus, in agreement with the X-ray data [2], histone H1 apparently is not required for maintenance of a compact state of DNA in chromatin subunits.     

Fluid-like chromatin: Toward understanding the real chromatin organization present in the cell

Eukaryotic chromatin is a negatively charged polymer consisting of genomic DNA, histones, and various nonhistone proteins. Because of its highly charged character, the structure of chromatin varies greatly depending on the surrounding environment (i.e. cations etc.): from an extended 10-nm fiber, to a folded 30-nm fiber, to chromatin condensates/liquid-droplets. Over the last ten years, newly developed technologies have drastically shifted our view on chromatin from a static regular structure to a more irregular and dynamic one, locally like a fluid. Since no single imaging (or genomics) method can tell us everything and beautiful images (or models) can fool our minds, comprehensive analyses based on many technical approaches are important to capture actual chromatin organization inside the cell. Here we critically discuss our current view on chromatin and methodology used to support the view.     

Dynamic and static chromatin

It was found that the dependence of the viscosity of calf thymus chromatin dispersions and human leukocytes on ethidium bromide concentration had two peaks indicative of domains with circular supercoiled DNA and varying resistance to ultrasound in the cells and isolated chromatin. The hypothesis of V. D. Paponov and P. S. Gromov (Bull. Exp. Biol. Med., N5, 590, 1985) on the transformation of static relations of nucleosome DNA-containing nuclei into dynamic, after chromatin exposure to ultrasound due to DNA linearization in chromatin domains possessing circular supercoiled DNA, has been confirmed.     

Roles of chromatin assembly factor 1 in the epigenetic control of chromatin plasticity

Genetic information embedded in DNA sequence and the epigenetic information marked by modifications on DNA and histones are essential for the life of eukaryotes. Cells have evolved mechanisms of DNA duplication and chromatin restoration to ensure the inheritance of genetic and epigenetic information during cell division and development. In this review, we focus on the maintenance of epigenetic landscape during chromatin dynamics which requires the orchestration of histones and their chaperones. We discuss how epigenetic marks are re-established in the assembly of new chromatin after DNA replication and repair, highlighting the roles of CAF-1 in the process of changing chromatin state. The functions of CAF-1 provide a link between chromatin assembly and epigenetic restoration.     

Spectroscopic study of fast-neutron-irradiated chromatin

The effects produced by fast neutrons (0-100 Gy) on chromatin structure were analyzed by (i) [1H]-NMR spectroscopy, (ii) time resolved spectroscopy, and (iii) fluorescence resonance energy transfer (FRET). Two types of chromatin were tested: (i) a chromatin from a normal tissue (liver of Wistar rats) and (ii) a chromatin from a tumoral tissue (Guerin limphotrope epithelioma, a rat solid tumor). The fast-neutron action on chromatin determines greater values of the [1H]-NMR transverse relaxation time, indicating a more injured structure. Time-resolved fluorescence measurements show that the relative contribution of the excited state lifetime of bound ethidium bromide to chromatin DNA diminishes with increasing irradiation doses. This reflects the damage that occurs in DNA structure: production of single- and double-strand breaks due to sugar and base modifications. By the FRET method, the distance between dansyl chloride and acridine orange coupled at chromatin was determined. This distance increases upon fast-neutron action. The radiosensitivity of the tumor tissue chromatin seems higher than that of the normal tissue chromatin, probably because of its higher (loose) euchromatin/(compact) heterochromatin ratio. As the values of the physical parameters analyzed are specific for a determined dose, the establishment of these parameters may constitute a criterion for the microdosimetry of chromatin radiolesions produced by fast neutrons.     

Unconventional metabolites in chromatin regulation

Chromatin, the complex of DNA and histone proteins, serves as a main integrator of cellular signals. Increasing evidence links cellular functional to chromatin state. Indeed, different metabolites are emerging as modulators of chromatin function and structure. Alterations in chromatin state are decisive for regulating all aspects of genome function and ultimately have the potential to produce phenotypic changes. Several metabolites such as acetyl-CoA, S-adenosylmethionine (SAM) or adenosine triphosphate (ATP) have now been well characterized as main substrates or cofactors of chromatin-modifying enzymes. However, there are other metabolites that can directly interact with chromatin influencing its state or that modulate the properties of chromatin regulatory factors. Also, there is a growing list of atypical enzymatic and nonenzymatic chromatin modifications that originate from different cellular pathways that have not been in the limelight of chromatin research. Here, we summarize different properties and functions of uncommon regulatory molecules originating from intermediate metabolism of lipids, carbohydrates and amino acids. Based on the various modes of action on chromatin and the plethora of putative, so far not described chromatin-regulating metabolites, we propose that there are more links between cellular functional state and chromatin regulation to be discovered. We hypothesize that these connections could provide interesting starting points for interfering with cellular epigenetic states at a molecular level.     

Effects of shearing on chromatin structure

The effects of mechanical shearing on chromatin structure were investigated by using thermal denaturation and circular dichroism (CD) spectroscopy. Under ordinary conditions of mechanical shearing used for preparation of soluble chromatin, we observed only minor changes (less than 10%) of chromatin properties with respect to (a) absorption melting curves, (b) CD spectra, (c) CD melting curves and (d) histone transfer from chromatin to exogenous DNA. Such small pertubation of structural properties could be due to the generation of free ends when a large chromatin was cut into smaller fragments and by weakening the binding of histones to DNA near these free ends. In addition to mechanical shearing, sonication was used to shear some samples of chromatin. The effect of sonication on chromatin structure was investigated by the same physical methods used for mechanically sheared chromatin. The results indicate that sonication only slightly changes the chromatin properties with respect to CD spectra, similar to the results obtained by mechanical shearing, but sonication at high settings has a greater effect on the thermal denaturation property of chromatin as contrasted to our results from mechanically sheared chromatin.     

Relationship between chromatin compactness and dye uptake for in situ chromatin stained with DAPI

BACKGROUND: This study investigated the relationship between chromatin compactness, which is directly related to chromatin condensation, and DAPI uptake. Materials and Methods For the structural characterization of in situ chromatin, we used fluorescence microscopy and differential scanning calorimetry on calf thymocytes. The compactness of nuclear chromatin was altered by permeabilizing native cells with NP40 detergent. A time-dependent analysis of detergent effects was performed by acquiring nuclear images at different time intervals after permeabilization. In order to compare nuclei of different sizes, we implemented a geometrical correction in the calculation of the integrated fluorescence intensity. For a quantitative evaluation of chromatin condensation we introduced two new parameters, "average chromatin packing ratio" and "average dye spatial density." RESULTS: This approach allowed us to estimate the effects of NP40 detergent at the level of in situ chromatin. Detergent effects could be modulated by changing the ionic composition of buffer. Moreover, changes of chromatin condensation induced by detergent were inversely related to modifications of nuclear volume. CONCLUSIONS: The combination of complementary information obtained by fluorescence microscopy, supported by a proper geometrical correction, and differential calorimetry allowed us to interpret the patterns of fluorescence intensities inside the nucleus in terms of chromatin structure.