Characterization of chromatin samples in the presence of Drosophila embryo extract by quantitative agarose gel electrophoresis

Recent studies have focused attention on chromatin as both a negative and positive regulator of specific nuclear events. The vast majority of this research has been centered on chromatin remodeling and histone post-translational modifications over the regulatory regions of specific genes. However, due the technical difficulties of such studies, the contribution of the higher-order structure of chromatin on the regulation of gene expression has not been as thoroughly investigated and the majority of the initial studies have used biophysical methods or microscopy. Until recent technical developments, the main hindrance for these biophysical investigations of chromatin has been an almost absolute requirement for large amounts of highly purified material. The development of an agarose gel electrophoresis method (quantitative agarose gel electrophoresis), initially designed for the analysis of the three-dimensional structure of purified and in vivo-assembled chromatin over a promoter region, has been expanded to include studies of chromatin in the presence of a Drosophila crude extract. The technique presented in the study reported here will help in paving the way for subsequent analyses of structural modifications of chromatin that are linked with the recruitment of various chromatin-associated factors present in the provided extract(s).     

Changes of chromatin condensation in one patient with ataxia telangiectasia disorder: a structural study

Differential scanning calorimetry and quantitative fluorescence microscopy have been employed to characterize the structure and organization of in situ chromatin in lymphoblastoid cells obtained from one ataxia telangiectasia (A-T) patient and one healthy family member. The proven capability of these biophysical techniques to measure changes of chromatin condensation directly inside the cells makes them very powerful in studying the eventual structural changes associated with the appearance of a pleiotropic genetic disorder such as ataxia telangiectasia. A-T syndrome is genetically heterogeneous and can be induced by different mutations of a single gene. The aim of this work is to determine whether the genetic mutation exhibited by the A-T patient of this study may be associated with modifications of chromatin structure and organization. Both the calorimetric and the fluorescence microscopy results acquired on cells from the A-T patient show that the structure and distribution of nuclear chromatin in situ change considerably with respect to the control. A significant decondensation of the nuclear chromatin is in fact associated with the appearance of the A-T disorder in the A-T patient under analysis, together with a rearrangement of the chromatin domains inside the nucleus.     

The Force is Strong with This Epigenome: Chromatin Structure and Mechanobiology

All life forms sense and respond to mechanical stimuli. Throughout evolution, organisms develop diverse mechanosensing and mechanotransduction pathways, leading to fast and sustained mechanoresponses. Memory and plasticity characteristics of mechanoresponses are thought to be stored in the form of epigenetic modifications, including chromatin structure alterations. These mechanoresponses in the chromatin context share conserved principles across species, such as lateral inhibition during organogenesis and development. However, it remains unclear how mechanotransduction mechanisms alter chromatin structure for specific cellular functions, and if altered chromatin structure can mechanically affect the environment. In this review, we discuss how chromatin structure is altered by environmental forces via an outside-in pathway for cellular functions, and the emerging concept of how chromatin structure alterations can mechanically affect nuclear, cellular, and extracellular environments. This bidirectional mechanical feedback between chromatin of the cell and the environment can potentially have important physiological implications, such as in centromeric chromatin regulation of mechanobiology in mitosis, or in tumor-stroma interactions. Finally, we highlight the current challenges and open questions in the field and provide perspectives for future research.     

Der Zellkern – eine Stadt in der Zelle,Teil 2

The Cell Nucleus – A Town in the Cell. Part II: Chromatin Nanoarchitecture and Gene Regulation In the second part of this article on nuclear architecture we describe the ground‐breaking potential of super‐resolution fluorescence microscopy (nanoscopy) and new molecular approaches to study the structure of chromatin domains at the nanometer scale and explore their 3D positions in larger chromatin domain clusters. New results and models argue for a decisive role of these structures in nuclear functions, such as gene regulation.     

动物早期胚胎发育中染色质结构的继承和重编程

染色质开放性和染色质三维高级结构在基因表达和调控中发挥着非常重要的作用,广泛参与分化、发育、肿瘤发生等细胞生理过程,是表观遗传研究的热点领域之一。动物胚胎发育起始于终端分化的卵子受精形成全能性的受精卵。在精卵结合的过程中,染色质开放性和染色质三维高级结构发生了剧烈的变化,经历继承、重编程、重新建立的过程,并指导调控受精卵分化发育最终成为多细胞、多器官组织的新生命个体。本文介绍了近年来研究染色质开放性和染色质三维高级结构的实验分析技术手段,染色质结构在动物早期胚胎发育过程中的变化规律及其在早期胚胎发育中的作用,染色质结构与其他表观遗传信息(甲基化、组蛋白修饰等)关系方面的重要研究进展和存在的科学问题,以期为表观遗传调控早期胚胎发育的研究提供参考。     

The role of chromatin proteins in DNA damage recognition and repair Mini-review

The structure of chromatin is the major factor determining the rate and efficiency of DNA repair. Chromatin remodeling events such as rearrangement of nucleosomes and higher order chromatin structures are indispensable features of repair processes. During the last decade numerous chromatin proteins have been identified that preferentially bind to different types of DNA damage. The HMGB proteins, which preferentially interact with DNA intrastrand crosslinks induced by cisplatin, are the archetypal example of such proteins. Several hypothetical models have been proposed describing the role of such damage-binding chromatin proteins. The damage shielding model postulates that binding of chromatin proteins to damaged DNA might disturb damage recognition by repair factors and impair its removal. Alternatively, the damage-recognition/signaling model proposes that the binding of specific chromatin proteins to damaged DNA could serve as a hallmark to be recognized by repair proteins. Additionally, the binding of specific chromatin proteins to damaged DNA could induce chromatin remodeling at the damage site and indirectly affect its repair. This paper aims to critically review current experimental data in relation to such possible roles of chromatin proteins.     

Chromatin structure as a mediator of aging

The aging process is characterized by gradual changes to an organism’s macromolecules, which negatively impacts biological processes. The complex macromolecular structure of chromatin regulates all nuclear processes requiring access to the DNA sequence. As such, maintenance of chromatin structure is an integral component to deter premature aging. In this review, we describe current research that links aging to chromatin structure. Histone modifications influence chromatin compaction and gene expression and undergo many changes during aging. Histone protein levels also decline during aging, dramatically affecting chromatin structure. Excitingly, lifespan can be extended by manipulations that reverse the age-dependent changes to chromatin structure, indicating the pivotal role chromatin structure plays during aging.