The p400 ATPase regulates nucleosome stability and chromatin ubiquitination during DNA repair

The complexity of chromatin architecture presents a significant barrier to the ability of the DNA repair machinery to access and repair DNA double-strand breaks (DSBs). Consequently, remodeling of the chromatin landscape adjacent to DSBs is vital for efficient DNA repair. Here, we demonstrate that DNA damage destabilizes nucleosomes within chromatin regions that correspond to the γ-H2AX domains surrounding DSBs. This nucleosome destabilization is an active process requiring the ATPase activity of the p400 SWI/SNF ATPase and histone acetylation by the Tip60 acetyltransferase. p400 is recruited to DSBs by a mechanism that is independent of ATM but requires mdc1. Further, the destabilization of nucleosomes by p400 is required for the RNF8-dependent ubiquitination of chromatin, and for the subsequent recruitment of brca1 and 53BP1 to DSBs. These results identify p400 as a novel DNA damage response protein and demonstrate that p400-mediated alterations in nucleosome and chromatin structure promote both chromatin ubiquitination and the accumulation of brca1 and 53BP1 at sites of DNA damage.     

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.     

Mechanical model of the nucleosome and chromatin

A theoretical framework for evaluating the approximate energy and dynamic properties associated with the folding of DNA into nucleosomes and chromatin is presented. Experimentally determined elastic constants of linear DNA and a simple fold geometry are assumed in order to derive elastic constants for extended and condensed chromatin. The model predicts the Young s modulus of extended and condensed chromatin to within an order of magnitude of experimentally determined values. Thus we demonstrate that the elastic properties of DNA are a primary determinant of the elastic properties of the higher order folded states. The derived elastic constants are used to predict the speed of propagation of small amplitude waves that excite an extension(sound), twist, bend or shear motion in each folded state. Taken together the results demonstrate that folding creates a hierarchy of time, length and energy scales.     

Novel function of PERK as a mediator of force-induced apoptosis

Induction of apoptosis by tensile forces is an important determinant of connective tissue destruction in osteoarthritis and periodontal diseases. We examined the role of molecular components of the unfolded protein response in force-induced apoptosis. Magnetic fields were used to apply tensile force through integrins to cultured fibroblasts bound with collagen-coated magnetite beads. Tensile force induced caspase 3 cleavage, DNA fragmentation, depolarization of mitochondria, and induction of CHOP10, all indicative of activation of apoptosis. Immunoblotting, immunocytochemistry, and release of Ca(2+) from the endoplasmic reticulum showed evidence for both physical and functional associations between bound beads and the endoplasmic reticulum. Force-induced apoptosis was not detected in PERK null cells, but reconstitution of wild-type PERK in PERK null cells restored the apoptotic response. Force-induced apoptosis did not require PKR, GCN2, eIF2alpha, or CHOP10. Furthermore, force more than 24 h did not activate other initiators of the unfolded protein response including IRE-1 and ATF6. However, force-induced activation of caspase 3 was dependent on caspase 9 but was independent of mitochondria. We conclude that force-induced apoptosis depends on a novel function of PERK that occurs in addition to its canonical role in the unfolded protein response.     

Nuclease digestion patterns as a criterion for nucleosome orientation in the higher order structure of chromatin

70 S ribosomes were programmed with initiator tRNA and messenger oligonucleotides AUG(U)n and AUG(C)n, where n = 1, 2 or 3. The binding of the ternary complexes [Phe-tRNA X EF-Tu X GTP] and [Pro-tRNA X EF-Tu X GTP] to the programmed ribosomes was studied. If codon-anticodon interaction is restricted to only one basepair, the ternary complex leaves the ribosome before GTP hydrolysis. Two basepairs allow hydrolysis of GTP, but the aminoacyl-tRNA dissociates and is recycled, resulting in wastage of GTP. Three basepairs result in apparently stable binding of aminoacyl-tRNA to the ribosome. The antibiotic sparsomycin weakens the binding by an amount roughly equivalent to one messenger base, while viomycin has the reverse effect.     

Single nucleosome tracking to study chromatin plasticity

The dynamic spatial organization of chromatin within the nucleus is emerging as a key regulator of gene activity and cell phenotype. This review will focus on single molecule tracking as an enabling tool to study chromatin dynamics at the level of individual nucleosomes.     

Spontaneous release of nucleosome cores from embryoblast chromatin

During in vitro incubation of the rabbit blastocyst, chromatin fragments of the nucleosome series are released spontaneously by cells of the pluripotent embryoblast but not by the developmentally restricted trophoblast. The DNA of these fragments is double stranded and linear, and represents total genomic DNA. The fragments are stable for at least 24 hr and are initially found in the cytoplasm of intact cells. The kinetics of radiolabeling of the DNA component of these fragments suggest that they originate from actively replicating cells, possibly only at the time of mitosis. DNase II-like activity, one of the endonucleases known preferentially to attack spacer DNA between nucleosome cores, is readily detectable in the blastocyst, although the evidence for its involvement in the observed chromatin fragmentation remains circumstantial. These nonrandom chromatin breaks, presumably representing an early event in cell death, occur more frequently under suboptimal conditions of incubation but are still detectable in embryos developing normally. There is no evidence for lysosomal activation in these dying cells. These findings, together with previous reports of identical chromatin fragmentation in other cell systems, suggest that this form of cell death in the embryoblast may be only one possible result of an active process, and that the mechanisms responsible for generating double-strand breaks in internucleosomal DNA may be of more biological significance than DNA degradation alone.     

Genome-wide nucleosome specificity and directionality of chromatin remodelers

How chromatin remodelers cooperate to organize nucleosomes around the start and end of genes is not known. We determined the genome-wide binding of remodeler complexes SWI/SNF, RSC, ISW1a, ISW1b, ISW2, and INO80 to individual nucleosomes in Saccharomyces, and determined their functional contributions to nucleosome positioning through deletion analysis. We applied ultra-high-resolution ChIP-exo mapping to Isw2 to determine its subnucleosomal orientation and organization on a genomic scale. Remodelers interacted with selected nucleosome positions relative to the start and end of genes and produced net directionality in moving nucleosomes either away or toward nucleosome-free regions at the 5' and 3' ends of genes. Isw2 possessed a subnucleosomal organization in accord with biochemical and crystallographic-based models that place its linker binding region within promoters and abutted against Reb1-bound locations. Together, these findings reveal a coordinated position-specific approach taken by remodelers to organize genic nucleosomes into arrays.     

Higher-order structure of nucleosome oligomers from short-repeat chromatin

Sedimentation measurements and electron microscopy at a series of ionic strengths suggest that chromatin from neurons of the cerebral cortex is able to form condensed structures in vitro that are probably several turns of a solenoid with about six nucleosomes per turn. Since neuronal chromatin has a short nucleosomal repeat (approximately 165 bp) allowing virtually no linker DNA between nucleosomes, and yet forms apparently 'normal' elements of solenoid, the packing of nucleosomes in the solenoid must be highly constrained. This permits only a limited number of possible models, and enables tentative suggestions to be made about the location of the linker DNA in the typical solenoid.     

DNA structure, nucleosome placement and chromatin remodelling: a perspective

A major question in chromatin biology is to what extent the sequence of DNA directly determines the genetic and chromatin organization of a eukaryotic genome? We consider two aspects to this question: the DNA sequence-specified positioning of nucleosomes and the determination of NDRs (nucleosome-depleted regions) or barriers. We argue that, in budding yeast, while DNA sequence-specified nucleosome positioning may contribute to positions flanking the regions lacking nucleosomes, DNA thermodynamic stability is a major component determinant of the genetic organization of this organism.     

Energy-dependent chromatin accessibility and nucleosome mobility in a cell-free system.

Chromatin structure must be flexible to allow the binding of regulatory proteins and to accommodate different levels of gene activity. Chromatin assembled in a cell-free system derived from Drosophila embryos contains an activity that hydrolyses ATP to render entire nucleosome arrays mobile. Nucleosome movements, most likely their sliding, occurred even in the presence of the linker histone H1. The dynamic state of chromatin in the presence of the activity and ATP globally increased the accessibility of nucleosomal DNA to incoming proteins. This unprecedented demonstration of energy-dependent nucleosome mobility identifies a new principle which is likely to be fundamental to the mechanism of chromatin remodelling and the binding of regulatory proteins.     

Dynamic chromatin: concerted nucleosome remodelling and acetylation

The flexibility of chromatin that enables translation of environmental cues into changes in genome utilisation, relies on a battery of enzymes able to modulate chromatin structure in a highly targeted and regulated manner. The most dynamic structural changes are brought about by two kinds of enzymes with different functional principles. Changes in the acetylation status of histones modulate the folding of the nucleosomal fibre. The histone-DNA interactions that define the nucleosome itself can be disrupted by ATP-dependent remodelling factors. This review focuses on recent developments that illustrate various strategies for integrating these disparate activities into complex regulatory schemes. Synergies may be brought about by consecutive or parallel action during the stepwise process of chromatin opening or closing. Tight co-ordination may be achieved by direct interaction of (de-)acetylation enzymes and remodelling ATPases or even permanent residence within the same multi-enzyme complex. The fact that remodelling ATPases can be acetylated by histone acetyltransferases themselves suggests exciting possibilities for the co-ordinate modulation of chromatin structure and remodelling enzymes.     

Nascent DNA in nucleosome like structures from chromatin

We have used chromatin sensitivity to cleavage by micrococcal nuclease as a probe for differences between chromatin containing nascent DNA and that containing bulk DNA. Micrococcal nuclease digested the nascent DNA in chromatin of swimming blastulae of sea urchins more rapidly to acid-soluble nucleotides than the DNA of bulk chromatin. A part of the nascent DNA occurred in micrococcal nuclease-resistant structures which were either different from, or temporary modifications of, the bulk nucleosomes. This was inferred from the size differences between bulk and nascent DNA fragments in 10% polyacrylamide gels after micrococcal nuclease digestion of nuclei from a mixture of ¹⁴C-thymidine long- and ³H-thymidine pulse-labeled embryos. Bulk monomer and dimer DNA fragments contained about 170 and 410 base pairs (bp), respectively, when 18% of the bulk DNA had been rendered acid-soluble. At this level of digestion, “nascent monomer DNA” fragments of about 150 bp as well as 305 bp “large nascent DNA fragments” were observed. Increasing levels of digestion indicated that the large nascent DNA fragment was derived from a chromatin structure which was more resistant to micrococcal nuclease cleavage than bulk dimer chromatin subunits. Peaks of ³H-thymidine-labeled DNA fragments from embryos which had been pulse-labeled and then chased or labeled for several minutes overlapped those of ¹⁴C-thymidine long-labeled monomer, dimer and trimer fragments. This indicated that the chromatin organization at or near the replication fork which had temporarily changed during replication had returned to the organization of its nonreplicating state.