DNA instructed displacement of histones H2A and H2B at an inducible promoter

Regulation of gene expression requires dynamic changes in chromatin, but the nature of these changes is not well understood. Here, we show that progesterone treatment of cultured cells leads to recruitment of progesterone receptor (PR) and SWI/SNF-related complexes to Mouse Mammary Tumor Virus (MMTV) promoter, accompanied by displacement of histones H2A and H2B from the nucleosome containing the receptor binding sites, but not from adjacent nucleosomes. PR recruits SWI/SNF to MMTV nucleosomes in vitro and facilitates synergistic binding of receptors and nuclear factor 1 to the promoter. In nucleosomes assembled on MMTV or mouse rDNA promoter sequences, SWI/SNF catalyzes ATP-dependent sliding of the histone octamer followed only on the MMTV promoter by displacement of histones H2A and H2B. In MMTV nucleosome arrays, SWI/SNF displaces H2A and H2B from nucleosome B and not from the adjacent nucleosome. Thus, the outcome of nucleosome remodeling by SWI/SNF depends on DNA sequence.     

ATP-dependent chromatin remodeling enzymes: two heads are not better, just different

ATP-dependent chromatin remodeling complexes enable rapid rearrangements in chromatin structure in response to developmental cues. The ATPase subunits of remodeling complexes share homology with the helicase motifs of DExx box helicases. Recent single-molecule experiments indicate that, like helicases, many of these complexes use ATP to translocate on DNA. Despite sharing this fundamental property, two key classes of remodeling complexes, the ISWI class and the SWI/SNF class, generate distinct remodeled products. SWI/SNF complexes generate nucleosomes with altered positions, nucleosomes with DNA loops and nucleosomes that are capable of exchanging histone dimers or octamers. In contrast, ISWI complexes generate nucleosomes with altered positions but in standard structures. Here, we draw analogies to monomeric and dimeric helicases and propose that ISWI and SWI/SNF complexes catalyze different outcomes in part because some ISWI complexes function as dimers while SWI/SNF complexes function as monomers.     

Antagonistic forces that position nucleosomes in vivo

ATP-dependent chromatin remodeling complexes are implicated in many areas of chromosome biology. However, the physiological role of many of these enzymes is still unclear. In budding yeast, the Isw2 complex slides nucleosomes along DNA. By analyzing the native chromatin structure of Isw2 targets, we have found that nucleosomes adopt default, DNA-directed positions when ISW2 is deleted. We provide evidence that Isw2 targets contain DNA sequences that are inhibitory to nucleosome formation and that these sequences facilitate the formation of nuclease-accessible open chromatin in the absence of Isw2. Our data show that the biological function of Isw2 is to position nucleosomes onto unfavorable DNA. These results reveal that antagonistic forces of Isw2 and the DNA sequence can control nucleosome positioning and genomic access in vivo.     

Generation of superhelical torsion by ATP-dependent chromatin remodeling activities

ATP-dependent chromatin remodeling activities participate in the alteration of chromatin structure during gene regulation. All have DNA- or chromatin-stimulated ATPase activity and many can alter the structure of chromatin; however, the means by which they do this have remained unclear. Here we describe a novel activity for ATP-dependent chromatin remodeling activities, the ability to generate unconstrained negative superhelical torsion in DNA and chromatin. We find that the ability to distort DNA is shared by the yeast SWI/SNF complex, Xenopus Mi-2 complex, recombinant ISWI, and recombinant BRG1, suggesting that the generation of superhelical torsion represents a primary biomechanical activity shared by all Snf2p-related ATPase motors. The generation of superhelical torque provides a potent means by which ATP-dependent chromatin remodeling activities can manipulate chromatin structure.     

AFM imaging of protein movements: histone H2A-H2B release during nucleosome remodeling

Being able to follow assembly/disassembly reactions of biomolecular complexes directly at the single molecule level would be very useful. Here, we use an AFM technique that can simultaneously obtain topographic images and identify the locations of a specific type of protein within those images to monitor the histone H2A component of nucleosomes acted on by human Swi-Snf, an ATP-dependent nucleosome remodeling complex. Activation of remodeling results in significant H2A release from nucleosomes, based on recognition imaging and nucleosome height changes, and changes in the recognition patterns of H2A associated directly with hSwi-Snf complexes.     

Human SWI/SNF nucleosome remodeling activity is partially inhibited by linker histone H1

The physical structure and the compact nature of the eukaryotic genome present a functional barrier for any cellular process that requires access to the DNA. The linker histone H1 is intrinsically involved in both the determination of and the stability of higher order chromatin structure. Because histone H1 plays a pivotal role in the structure of chromatin, we investigated the effect of histone H1 on the nucleosome remodeling activity of human SWI/SNF, an ATP-dependent chromatin remodeling complex. The results from both DNase I digestion and restriction endonuclease accessibility assays indicate that the presence of H1 partially inhibits the nucleosome remodeling activity of hSWI/SNF. Neither H1 bound to the nucleosome nor free H1 affected the ATPase activity of hSWI/SNF, suggesting that the observed inhibition of hSWI/SNF nucleosome remodeling activity depends on the structure formed by the addition of H1 to nucleosomes.     

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。     

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.     

Particular structural role of H1 in complexes with DNA and comparison with H2A- and H4-DNA complexes investigated by IR linear dichroism.

The particular role of H1 in the structure of histone–DNA associations is shown by means of ir linear dichroism. H1–, H2A–, and H4–DNA complexes are studied for different histone: DNA input ratios and various relative humidities (r.h.). The measurement of the dichroic ratios allows one to determine the secondary structure of DNA in the complexes. It is shown that the progressive addition of histone H2A or H4 to DNA inhibits the structural B → A transition and DNA remains in a B-type form at low r.h. It is found that the B → A transition is inhibited for 19 or 26 base pairs of DNA per molecule of H2A or H4. The stabilization of DNA in a B-conformation by H2A and H4 has been also observed by H2B and H3 but with a different efficiency. In contrast, histone H1, which does not belong to the core of the nucleosomes in chromatin, leaves the DNA in H1–DNA complexes free to adopt an A conformation at low r.h. for H1/DNA ratios below 0.6/1. Thus a major difference in the structural role between histone H1 and histones belonging to the nucleosomal core with respect to the conformational flexibility of DNA in the histone–DNA complexes is demonstrated.     

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.     

Chromatin Remodelers Act Globally, Sequence Positions Nucleosomes Locally

The precise placement of nucleosomes has large regulatory effects on gene expression. Recent work suggests that nucleosome placement is regulated in part by the affinity of the underlying DNA sequence for the histone octamer. Nucleosome locations are also regulated by several different ATP-dependent chromatin remodeling enzymes. This raises the question of whether DNA sequence influences the activity of chromatin remodeling enzymes. DNA sequence could most simply regulate nucleosome remodeling through its effect on nucleosome stability. In such a model, unstable nucleosomes would be remodeled faster than stable nucleosomes. It is also possible that certain DNA elements could regulate remodeling by inhibiting the interaction of nucleosomes with the remodeling enzyme. A third possibility is that DNA sequence could regulate the outcome of remodeling by influencing how reaction intermediates collapse into a particular set of stable nucleosomal positions. Here we dissect the contribution from these potential mechanisms to the activities of yeast RSC and human ACF, which are representative members of two major classes of remodeling complexes. We find that varying the histone-DNA affinity over 3 orders of magnitude has negligible effects on the rates of nucleosome remodeling and ATP hydrolysis by these two enzymes. This suggests that the rate-limiting step for nucleosome remodeling may not involve the disruption of histone-DNA contacts. We further find that a specific curved DNA element previously hypothesized to inhibit ACF activity does not inhibit substrate binding or remodeling by ACF. The element, however, does influence the distribution of nucleosome positions generated by ACF. Our data support a model in which remodeling enzymes move nucleosomes to new locations by a general sequence-independent mechanism. However, consequent to the rate-limiting remodeling step, the local DNA sequence promotes a collapse of remodeling intermediates into highly resolved positions that are dictated by thermodynamic differences between adjacent positions.     

The INO80 complex is required for damage-induced recombination

The budding yeast INO80 complex has a role in remodeling chromatin structure, and the SWR1 complex replaces a H2A/H2B dimer with a variant dimer, H2A.Z (Htz1)/H2B. It has been reported that these chromatin remodeling complexes contain Arp4 (actin-related protein) and actin in common and are recruited to HO endonuclease-induced DNA double-strand break (DSB) site. Reportedly, Ino80 can evict nucleosomes surrounding a HO-induced DSB; however, it has no apparent role to play in the repair of HO-induced DSB. Here we show that an essential factor for INO80 chromatin remodeling activity, Arp8, is involved in damage-induced sister chromatid recombination and interchromosomal recombination between heteroalleles. In contrast, arp6 mutants are proficient for recombination, indicating that the SWR1 complex does not promote recombination. Our data suggest that the remodeling of chromatin by the INO80 complex facilitates efficient homologous recombination upon DNA damages.