Histone variants and their chaperones are key regulators of eukaryotic transcription, and are critical for normal development. The histone variant H3.3 has been shown to play important roles in pluripotency and differentiation, and although its genome-wide patterns have been investigated, little is known about the role of its dynamic turnover in transcriptional regulation. To elucidate the role of H3.3 dynamics in embryonic stem cell (ESC) biology, we generated mouse ESC lines carrying a single copy of a doxycycline (Dox)-inducible HA-tagged version of H3.3 and monitored the rate of H3.3 incorporation by ChIP-seq at varying time points following Dox induction, before and after RA-induced differentiation. Comparing H3.3 turnover profiles in ESCs and RA-treated cells, we identified a hyperdynamic H3.3-containing nucleosome at the −1 position in promoters of genes expressed in ESCs. This dynamic nucleosome is restricted and shifted downstream into the +1 position following differentiation. We suggest that histone turnover dynamics provides an additional mechanism involved in expression regulation, and that a hyperdynamic −1 nucleosome marks promoters in ESCs. Our data provide evidence for regional regulation of H3.3 turnover in ESC promoters, and calls for testing, in high resolution, the dynamic behavior of additional histone variants and other structural chromatin proteins.

Branched polymers can be represented as tree graphs. A one-to-one correspondence exists between a tree graph comprised of N labeled vertices and a sequence of N 2 integers, known as the Prufer sequence. Permutations of this sequence yield sequences corresponding to tree graphs with the same vertex-degree distribution but (generally) different branching patterns. Repeatedly shuffling the Prufer sequence we have generated large ensembles of random tree graphs, all with the same degree distributions. We also present and apply an efficient algorithm to determine graph distances directly from their Prufer sequences. From the (Prufer sequence derived) graph distances, 3D size metrics, e.g., the polymer’s radius of gyration, R-g, and average end-to-end distance, were then calculated using several different theoretical approaches. Applying our method to ideal randomly branched polymers of different vertex-degree distributions, all their 3D size measures are found to obey the usual N-1/4 scaling law. Among the branched polymers analyzed are RNA molecules comprised of equal proportions of the four-randomly distributed-nucleotides. Prior to Prufer shuffling, the vertices of their representative tree graphs, these ‘‘random-sequence’’ RNAs exhibit an R-g similar to N-1/3 scaling.

Long RNA molecules are at the core of gene regulation across all kingdoms of life, while also serving as genomes in RNA viruses. Few studies have addressed the basic physical properties of long single-stranded RNAs. Long RNAs with non repeating sequences usually adopt highly ramified secondary structures and are better described as branched polymers. To test whether a branched polymer model can estimate the overall sizes of large RNAs, we employed fluorescence correlation spectroscopy to examine the hydrodynamic radii of a broad spectrum of biologically important RNAs, ranging from viral genomes to long noncoding regulatory RNAs. The relative sizes of long RNAs measured at low ionic strength correspond well to those predicted by two theoretical approaches that treat the effective branching associated with secondary structure formation one employing the Kramers theorem for calculating radii of gyration, and the other featuring the metric of maximum ladder distance. Upon addition of multivalent cations, most RNAs are found to be compacted as compared with their original, low ionic-strength sizes. These results suggest that sizes of long RNA molecules are determined by the branching pattern of their secondary structures. We also experimentally validate the proposed computational approaches for estimating hydrodynamic radii of single stranded RNAs, which use generic RNA structure prediction tools and thus can be universally applied to a wide range of long RNAs. http://www.sciencedirect.com/science/article/pii/S0006349516309419

Aneuploidy is a hallmark of cancer and underlies genetic disorders characterized by severe developmental defects, yet the molecular mechanisms explaining its effects on cellular physiology remain elusive. Here we show, using a series of human cells with defined aneuploid karyotypes, that gain of a single chromosome increases genomic instability. Next-generation sequencing and SNP-array analysis reveal accumulation of chromosomal rearrangements in aneuploids, with break point junction patterns suggestive of replication defects. Trisomic and tetrasomic cells also show increased DNA damage and sensitivity to replication stress. Strikingly, we find that aneuploidy-induced genomic instability can be explained by the reduced expression of the replicative helicase MCM2-7. Accordingly, restoring near-wild-type levels of chromatin-bound MCM helicase partly rescues the genomic instability phenotypes. Thus, gain of chromosomes triggers replication stress, thereby promoting genomic instability and possibly contributing to tumorigenesis.

Common fragile sites (CFSs) are chromosomal regions characterized as hotspots for breakage and chromosomal rearrangements following DNA replication stress. They are preferentially unstable in pre-cancerous lesions and during cancer development. Recently CFSs were found to be tissue- and even oncogene-induced specific, thus indicating an unforeseen complexity. Here we review recent developments in CFS research that shed new light on the molecular basis of their instability and their importance in cancer development.

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To optimize binding-and packaging-by their capsid proteins (CP), single-stranded (ss) RNA viral genomes often have local secondary/tertiary structures with high CP affinity, with these ‘‘packaging signals’’ serving as heterogeneous nucleation sites for the formation of capsids. Under typical in vitro self-assembly conditions, however, and in particular for the case of many ssRNA viruses whose CP have cationic N-termini, the adsorption of CP by RNA is nonspecific because the CP concentration exceeds the largest dissociation constant for CP RNA binding. Consequently, the RNA is saturated by bound protein before lateral interactions between CP drive the homogeneous nucleation of capsids. But, before capsids are formed, the binding of protein remains reversible and introduction of another RNA species with a different length and/or sequence is found experimentally to result in significant redistribution of protein. Here we argue that, for a given RNA mass, the sequence with the highest affinity for protein is the one with the most compact secondary structure arising from self-complementarity; similarly, a long RNA steals protein from an equal mass of shorter ones. In both cases, it is the lateral attractions between bound proteins that determines the relative CP affinities of the RNA templates, even though the individual binding sites are identical. We demonstrate this with Monte Carlo simulations, generalizing the Rosenbluth method for excludedvolume polymers to include branching of the polymers and their reversible binding by protein.

For many viruses, the packaging of a single-stranded RNA (ss-RNA) genome is spontaneous, driven by capsid protein-capsid protein (CP) and CP-RNA interactions. Furthermore, for some multipartite ss-RNA viruses, copackaging of two or more RNA molecules is a common strategy. Here we focus on RNA copackaging in vitro by using cowpea chlorotic mottle virus (CCMV) CP and an RNA molecule that is short (500 nucleotides (nts)) compared to the lengths (approximate to 3000 nts) packaged in wild-type virions. We show that the degree of cooperativity of virus assembly depends not only on the relative strength of the CP-CP and CP-RNA interactions but also on the RNA being short: a 500-nt RNA molecule cannot form a capsid by itself, so its packaging requires the aggregation of multiple CP-RNA complexes. By using fluorescence correlation spectroscopy (FCS), we show that at neutral pH and sufficiently low concentrations RNA and CP form complexes that are smaller than the wild-type capsid and that four 500-nt RNAs are packaged into virus-like particles (VLPs) only upon lowering the pH. Further, a variety of bulk-solution techniques confirm that fully ordered VLPs are formed only upon acidification. On the basis of these results, we argue that the observed high degree of cooperativity involves equilibrium between multiple CP/RNA complexes.

The comment by Stephen Harvey in this issue of the Biophysical Journal concludes with two statements regarding my recent letter about DNA packaging into viral capsids. Harvey agrees with my interpretation of the origin of the large confinement entropy predicted by the molecular-dynamics simulations of his group, and its sensitive dependence on the molecular parameters of their wormlike chain model of double-stranded DNA. On the other hand, he doubts my assertion that the confinement entropy is already included in the interstrand repulsion free energy derived from osmotic stress measurements, which constitutes the major contribution to the packaging free energy used in recent continuum theories of this process. Harvey suggests instead that the confinement entropy should be added to this free energy as a separate term (using, for instance, the method described in my letter). I will argue that this addition is redundant, and, in a brief discussion of continuum theories, will also discuss his comments as relates to the work of other researchers.

A majority of viruses are composed of long single-stranded genomic RNA molecules encapsulated by protein shells with diameters of just a few tens of nanometers. We examine the extent to which these viral RNAs have evolved to be physically compact molecules to facilitate encapsulation. Measurements of equal-length viral, non-viral, coding and non-coding RNAs show viral RNAs to have among the smallest sizes in solution, i.e., the highest gel-electrophoretic mobilities and the smallest hydrodynamic radii. Using graph-theoretical analyses we demonstrate that their sizes correlate with the compactness of branching patterns in predicted secondary structure ensembles. The density of branching is determined by the number and relative positions of 3-helix junctions, and is highly sensitive to the presence of rare higher-order junctions with 4 or more helices. Compact branching arises from a preponderance of base pairing between nucleotides close to each other in the primary sequence. The density of branching represents a degree of freedom optimized by viral RNA genomes in response to the evolutionary pressure to be packaged reliably. Several families of viruses are analyzed to delineate the effects of capsid geometry, size and charge stabilization on the selective pressure for RNA compactness. Compact branching has important implications for RNA folding and viral assembly.