Biochemical Assays for Analyzing Activities of ATP-dependent Chromatin Remodeling Enzymes

Members of the SNF2 family of ATPases often function as components of multi-subunit chromatin remodeling complexes that regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Biochemically dissecting the contributions of individual subunits of such complexes to the multi-step ATP-dependent chromatin remodeling reaction requires the use of assays that monitor the production of reaction products and measure the formation of reaction intermediates. This JOVE protocol describes assays that allow one to measure the biochemical activities of chromatin remodeling complexes or subcomplexes containing various combinations of subunits. Chromatin remodeling is measured using an ATP-dependent nucleosome sliding assay, which monitors the movement of a nucleosome on a DNA molecule using an electrophoretic mobility shift assay (EMSA)-based method. Nucleosome binding activity is measured by monitoring the formation of remodeling complex-bound mononucleosomes using a similar EMSA-based method, and DNA- or nucleosome-dependent ATPase activity is assayed using thin layer chromatography (TLC) to measure the rate of conversion of ATP to ADP and phosphate in the presence of either DNA or nucleosomes. Using these assays, one can examine the functions of subunits of a chromatin remodeling complex by comparing the activities of the complete complex to those lacking one or more subunits. The human INO80 chromatin remodeling complex is used as an example; however, the methods described here can be adapted to the study of other chromatin remodeling complexes.     

Changing Chromatin Fiber Conformation by Nucleosome Repositioning

Chromatin conformation is dynamic and heterogeneous with respect to nucleosome positions, which can be changed by chromatin remodeling complexes in the cell. These molecular machines hydrolyze ATP to translocate or evict nucleosomes, and establish loci with regularly and more irregularly spaced nucleosomes as well as nucleosome-depleted regions. The impact of nucleosome repositioning on the three-dimensional chromatin structure is only poorly understood. Here, we address this issue by using a coarse-grained computer model of arrays of 101 nucleosomes considering several chromatin fiber models with and without linker histones, respectively. We investigated the folding of the chain in dependence of the position of the central nucleosome by changing the length of the adjacent linker DNA in basepair steps. We found in our simulations that these translocations had a strong effect on the shape and properties of chromatin fibers: i), Fiber curvature and flexibility at the center were largely increased and long-range contacts between distant nucleosomes on the chain were promoted. ii), The highest destabilization of the fiber conformation occurred for a nucleosome shifted by two basepairs from regular spacing, whereas effects of linker DNA changes of ∼10 bp in phase with the helical twist of DNA were minimal. iii), A fiber conformation can stabilize a regular spacing of nucleosomes inasmuch as favorable stacking interactions between nucleosomes are facilitated. This can oppose nucleosome translocations and increase the energetic costs for chromatin remodeling. Our computational modeling framework makes it possible to describe the conformational heterogeneity of chromatin in terms of nucleosome positions, and thus advances theoretical models toward a better understanding of how genome compaction and access are regulated within the cell.     

Changing Chromatin Fiber Conformation by Nucleosome Repositioning

Chromatin conformation is dynamic and heterogeneous with respect to nucleosome positions, which can be changed by chromatin remodeling complexes in the cell. These molecular machines hydrolyze ATP to translocate or evict nucleosomes, and establish loci with regularly and more irregularly spaced nucleosomes as well as nucleosome-depleted regions. The impact of nucleosome repositioning on the three-dimensional chromatin structure is only poorly understood. Here, we address this issue by using a coarse-grained computer model of arrays of 101 nucleosomes considering several chromatin fiber models with and without linker histones, respectively. We investigated the folding of the chain in dependence of the position of the central nucleosome by changing the length of the adjacent linker DNA in basepair steps. We found in our simulations that these translocations had a strong effect on the shape and properties of chromatin fibers: i), Fiber curvature and flexibility at the center were largely increased and long-range contacts between distant nucleosomes on the chain were promoted. ii), The highest destabilization of the fiber conformation occurred for a nucleosome shifted by two basepairs from regular spacing, whereas effects of linker DNA changes of ∼10 bp in phase with the helical twist of DNA were minimal. iii), A fiber conformation can stabilize a regular spacing of nucleosomes inasmuch as favorable stacking interactions between nucleosomes are facilitated. This can oppose nucleosome translocations and increase the energetic costs for chromatin remodeling. Our computational modeling framework makes it possible to describe the conformational heterogeneity of chromatin in terms of nucleosome positions, and thus advances theoretical models toward a better understanding of how genome compaction and access are regulated within the cell.     

Specialized RSC: Substrate Specificities for a Conserved Chromatin Remodeler

The remodel the structure of chromatin (RSC) nucleosome remodeling complex is a conserved chromatin regulator with roles in chromatin organization, especially over nucleosome depleted regions therefore functioning in gene expression. Recent reports in Saccharomyces cerevisiae have identified specificities in RSC activity toward certain types of nucleosomes. RSC has now been shown to preferentially evict nucleosomes containing the histone variant H2A.Z in vitro. Furthermore, biochemical activities of distinct RSC complexes has been found to differ when their nucleosome substrate is partially unraveled. Mammalian BAF complexes, the homologs of yeast RSC and SWI/SNF complexes, are also linked to nucleosomes with H2A.Z, but this relationship may be complex and extent of conservation remains to be determined. The interplay of remodelers with specific nucleosome substrates and regulation of remodeler outcomes by nucleosome composition are tantalizing questions given the wave of structural data emerging for RSC and other SWI/SNF family remodelers.     

染色质重塑因子在植物发育过程的功能

染色质重塑复合体(chromatin remodeling complexes)通过具有ATPase活性的亚基水解ATP释放能量,通过改变核小体"构象"(包括核小体重定位、核小体滑动和核小体替换等)而改变DNA的"可及性"(accessibility),进而影响特定的生理、生化过程。染色质重塑复合体最早在酵母中发现,生化分析表明其至少含有13个亚基。目前植物染色质重塑复合体的组成还未完全解析,但通过对其酵母同源亚基(染色质重塑因子)的研究可从侧面探究植物染色质重塑复合体的功能。同时,还着重讨论了近年来在植物染色质重塑因子研究上取得的结果,以期为植物染色质重塑的作用机制提供启示。     

On the role of inter-nucleosomal interactions and intrinsic nucleosome dynamics in chromatin function

Evidence is emerging that many diseases result from defects in gene functions, which, in turn, depend on the local chromatin environment of a gene. However, it still remains not fully clear how chromatin activity code is ‘translated’ to the particular ‘activating’ or ‘repressing’ chromatin structural transition. Commonly, chromatin remodeling in vitro was studied using mononucleosomes as a model. However, recent data suggest that structural reorganization of a single mononucleosome is not equal to remodeling of a nucleosome particle under multinucleosomal content – such as, interaction of nucleosomes via flexible histone termini could significantly alter the mode (and the resulting products) of nucleosome structural transitions. It is becoming evident that a nucleosome array does not constitute just a ‘polymer’ of individual ‘canonical’ nucleosomes due to multiple inter-nucleosomal interactions which affect nucleosome dynamics and structure. It could be hypothesized, that inter-nucleosomal interactions could act in cooperation with nucleosome inherent dynamics to orchestrate DNA-based processes and promote formation and stabilization of highly-dynamic, accessible structure of a nucleosome array. In the proposed paper we would like to discuss the nucleosome dynamics within the chromatin fiber mainly as it pertains to the roles of the structural changes mediated by inter-nucleosomal interactions.     

Telomeric nucleosomes are intrinsically mobile

Nucleosomes are no longer considered only static basic units that package eukaryotic DNA but they emerge as dynamic players in all chromosomal processes. Regulatory proteins can gain access to recognition sequences hidden by the histone octamer through the action of ATP-dependent chromatin remodeling complexes that cause nucleosome sliding. In addition, it is known that nucleosomes are able to spontaneously reposition along the DNA due to intrinsic dynamic properties, but it is not clear yet to what extent sequence-dependent dynamic properties contribute to nucleosome repositioning. Here, we study mobility of nucleosomes formed on telomeric sequences as a function of temperature and ionic strength. We find that telomeric nucleosomes are highly intrinsically mobile under physiological conditions, whereas nucleosomes formed on an average DNA sequence mostly remain in the initial position. This indicates that DNA sequence affects not only the thermodynamic stability and the positioning of nucleosomes but also their dynamic properties. Moreover, our findings suggest that the high mobility of telomeric nucleosomes may be relevant to the dynamics of telomeric chromatin.     

Methods for chromatin assembly and remodeling

Packaging of the DNA in nucleosomes restricts its accessibility to regulatory factors and enzymatic complexes, making a local remodeling of the nucleosome structure a prerequisite to the establishment of protein-DNA interactions. The use of an experimental system in which one nucleosome is reconstituted on a short linear DNA fragment allows gel fractionation of nucleosomes according to their translational positions, whose locations are dependent on the underlying DNA sequence. Nucleosome mobilization by chromatin remodeling factors is easily detected by observing band disappearance in gel, which in turn provides evidence for histone octamer displacement. Here, we provide methods for chromatin assembly that we have been using in our analysis for nucleosome mobilization by chromatin remodeling factors. These methods are straightforward and easy to follow. Thus, they may provide a good starting assay system for analysis of nucleosome movements by other chromatin remodeling machines.     

Chromatin remodeling activities act on UV-damaged nucleosomes and modulate DNA damage accessibility to photolyase

Nucleosomes inhibit DNA repair in vitro, suggesting that chromatin remodeling activities might be required for efficient repair in vivo. To investigate how structural and dynamic properties of nucleosomes affect damage recognition and processing, we investigated repair of UV lesions by photolyase on a nucleosome positioned at one end of a 226-bp-long DNA fragment. Repair was slow in the nucleosome but efficient outside. No disruption or movement of the nucleosome was observed after UV irradiation and during repair. However, incubation with the nucleosome remodeling complex SWI/SNF and ATP altered the conformation of nucleosomal DNA as judged by UV photo-footprinting and promoted more homogeneous repair. Incubation with yISW2 and ATP moved the nucleosome to a more central position, thereby altering the repair pattern. This is the first demonstration that two different chromatin remodeling complexes can act on UV-damaged nucleosomes and modulate repair. Similar activities might relieve the inhibitory effect of nucleosomes on DNA repair processes in living cells.     

Mechanisms of ATP dependent chromatin remodeling

The inter-relationship between DNA repair and ATP dependent chromatin remodeling has begun to become very apparent with recent discoveries. ATP dependent remodeling complexes mobilize nucleosomes along DNA, promote the exchange of histones, or completely displace nucleosomes from DNA. These remodeling complexes are often categorized based on the domain organization of their catalytic subunit. The biochemical properties and structural information of several of these remodeling complexes are reviewed. The different models for how these complexes are able to mobilize nucleosomes and alter nucleosome structure are presented incorporating several recent findings. Finally the role of histone tails and their respective modifications in ATP-dependent remodeling are discussed.     

Open, repair and close again: chromatin dynamics and the response to UV-induced DNA damage

Due to its link with human pathologies, including cancer, the mechanism of Nucleotide Excision Repair (NER) has been extensively studied. Most of the pathway and players have been defined using in vitro reconstitution experiments. However, in vivo, the NER machinery must deal with the presence of organized chromatin, which in some regions, such as heterochromatin, is highly condensed but still susceptible to DNA damage. A series of events involving different chromatin-remodeling factors and histone-modifying enzymes target chromatin regions that contain DNA lesions. CPDs change the structure of the nucleosome, allowing access to factors that can recognize the lesion. Next, DDB1-DDB2 protein complexes, which mono-ubiquitinate histones H2A, H3, and H4, recognize nucleosomes containing DNA lesions. The ubiquitinated nucleosome facilitates the recruitment of ATP-dependent chromatin-remodeling factors and the XPC-HR23B-Centrin 2 complex to the target region. Different ATP-dependent chromatin-remodeling factors, such as SWI/SNF and INO80, have been identified as having roles in the UV irradiation response prior to the action of the NER machinery. Subsequently, remodeling of the nucleosome allows enzymatic reactions by histone-modifying factors that may acetylate, methylate or demethylate specific histone residues. Intriguingly, some of these histone modifications are dependent on p53. These histone modifications and the remodeling of the nucleosome allow the entrance of TFIIH, XPC and other NER factors that remove the damaged strand; then, gap-filling DNA synthesis and ligation reactions are carried out after excision of the oligonucleotide with the lesion. Finally, after DNA repair, the initial chromatin structure has to be reestablished. Therefore, factors that modulate chromatin dynamics contribute to the NER mechanism, and they are significant in the future design of treatments for human pathologies related to genome instability and the appearance of drug-resistant tumors.     

ISWI is an ATP-dependent nucleosome remodeling factor

The ATPase ISWI is a subunit of several distinct nucleosome remodeling complexes that increase the accessibility of DNA in chromatin. We found that the isolated ISWI protein itself was able to carry out nucleosome remodeling, nucleosome rearrangement, and chromatin assembly reactions. The ATPase activity of ISWI was stimulated by nucleosomes but not by free DNA or free histones, indicating that ISWI recognizes a specific structural feature of nucleosomes. Nucleosome remodeling, therefore, does not require a functional interaction between ISWI and the other subunits of ISWI complexes. The role of proteins associated with ISWI may be to regulate the activity of the remodeling engine or to define the physiological context within which a nucleosome remodeling reaction occurs.