Active hippocampal networks undergo spontaneous synaptic modification

The brain is self-writable; as the brain voluntarily adapts itself to a changing environment, the neural circuitry rearranges its functional connectivity by referring to its own activity. How the internal activity modifies synaptic weights is largely unknown, however. Here we report that spontaneous activity causes complex reorganization of synaptic connectivity without any external (or artificial) stimuli. Under physiologically relevant ionic conditions, CA3 pyramidal cells in hippocampal slices displayed spontaneous spikes with bistable slow oscillations of membrane potential, alternating between the so-called UP and DOWN states. The generation of slow oscillations did not require fast synaptic transmission, but their patterns were coordinated by local circuit activity. In the course of generating spontaneous activity, individual neurons acquired bidirectional long-lasting synaptic modification. The spontaneous synaptic plasticity depended on a rise in intracellular calcium concentrations of postsynaptic cells, but not on NMDA receptor activity. The direction and amount of the plasticity varied depending on slow oscillation patterns and synapse locations, and thus, they were diverse in a network. Once this global synaptic refinement occurred, the same neurons now displayed different patterns of spontaneous activity, which in turn exhibited different levels of synaptic plasticity. Thus, active networks continuously update their internal states through ongoing synaptic plasticity. With computational simulations, we suggest that with this slow oscillation-induced plasticity, a recurrent network converges on a more specific state, compared to that with spike timing-dependent plasticity alone.     

Long-term cultured human neural stem cells undergo spontaneous transformation to tumor-initiating cells

In this report, we describe the spontaneous malignant transformation of long-term cultured human fetal striatum neural stem cells (hsNSCs, passage 17). After subcutaneous transplantation of long-term cultured hsNSCs into immunodeficient nude mice, 2 out of 15 mice formed xenografts which expressed neuroendocrine tumor markers CgA and NSE. T1 cells, a cell line that we derived from one of the two subcutaneous xenografts, have undergone continuous expansion in vitro. These T1 cells showed stem cell-like features and expressed neural stem cell markers nestin and CD133. The T1 cells were involved in abnormal karyotype, genomic instability and fast proliferation. Importantly, after long-term in vitro culture, the T1 cells did not result in subcutaneous xenografts, but induced intracranial tumor formation, indicating that they adjusted themselves to the intracranial microenvironment. We further found that the T1 cells exhibited an overexpressed level of EGFR, and the CD133 positive T1 cells showed a truncation mutation in the exons 2-7 of the EGFR (EGFRvIII) gene. These results suggest that continuous expansion of neural stem cells in culture may lead to malignant spontaneous transformation. This phenomenon may be functionally related to EGFR by EGFRvIII gene mutation.     

Variance and autocorrelation of the spontaneous slow brain activity

Slow (<0.1 Hz) oscillatory activity in the human brain, as measured by functional magnetic imaging, has been used to identify neural networks and their dysfunction in specific brain diseases. Its intrinsic properties may also be useful to investigate brain functions. We investigated the two functional maps: variance and first order autocorrelation coefficient (r(1)). These two maps had distinct spatial distributions and the values were significantly different among the subdivisions of the precuneus and posterior cingulate cortex that were identified in functional connectivity (FC) studies. The results reinforce the functional segregation of these subdivisions and indicate that the intrinsic properties of the slow brain activity have physiological relevance. Further, we propose a sample size (degree of freedom) correction when assessing the statistical significance of FC strength with r(1) values, which enables a better understanding of the network changes related to various brain diseases.     

Nucleosomes in the neighborhood

The importance of local chromatin structure in regulating replication initiation has become increasingly apparent. Most recently, histone methylation and nucleosome positioning have been added to the list of modifications demonstrated to regulate origins. In particular, the methylation states of H3K4, H3K36 and H4K20 have been associated with establishing active, repressed or poised origins depending on the timing and extent of methylation. The stability and precise positioning of nucleosomes has also been demonstrated to affect replication efficiency. Although it is not yet clear how these modifications alter the behavior of specific replication factors, ample evidence establishes their role in maintaining coordinated replication. This review will summarize recent advances in understanding these aspects of chromatin structure in DNA replication origin control.     

Nucleosomes arrangement in chromatin

The spatial arrangement of nucleosomes in rat liver chromatin has been examined using the electric birefringence technique. All chromatin subunits studied (up to 9 consecutive nucleosomes) contain their full complement of the five histone types associated with about 200 base pairs repeat length DNA.     

Nucleosomes facilitate their own invasion

DNA wrapped in nucleosomes is sterically occluded, creating obstacles for polymerase, regulatory, remodeling, repair and recombination complexes, which require access to the wrapped DNA. How such complexes recognize and gain access to their DNA target sites is not known. Here we report the direct detection of a dynamic equilibrium conformational transition in nucleosomes that greatly increases the distance between the end of the nucleosomal DNA and the histone core. We quantified the equilibrium constant for this transition under physiological conditions. As predicted by these findings, addition of LexA protein to nucleosomes containing the LexA target site drives this conformational equilibrium toward the unwrapped, accessible state, simultaneously allowing stable LexA binding. This inherent property of nucleosomes allows any protein, whether an energy-dependent machine or a passive binder, to gain access even to buried stretches of nucleosomal DNA.     

Nucleosomes in metaphase chromosomes.

Previous studies of the structure of metaphase chromosomes have relied heavily on electron micrography and have revealed the existence of a 10-nm unit fiber that is thought to generate the native 23-30-nm fiber by higher order folding. The structural relationship of these metaphase fibers to the interphase fiber remains obscure. Recent studies on the digestion of interphase chromatin have revealed the existence of a regularly repeating subunit of DNA and histone, the nucleosome that generates the appearance of 10-nm beads connected by a short fiber of DNA seen on electron micrographs. It was therefore of interest to probe the structure of the metaphase chromosome for the presence of nucleosomal subunits. To this end metaphase chromosomes were prepared from colchicine-arrested cultures of mouse L-cells and were subjected to digestion with stayphylococcal nuclease. Comparison of the early and limit digestion products of metaphase chromosomes with those obtained from interphase nuclei indicates that although significant morphologic changes occur within the chromatin fiber during mitosis, the basic subunit structure of the chromatin fiber is retained by the mitotic chromosome.     

Mesenchymal stem cells from human endometrium do not undergo spontaneous transformation during long-term cultivation

Human-endometrium mesenchymal stem cells (eMSCs) are a promising source of stem cells for regenerative medicine. A large amount of these cells accumulated by in vitro cultivation are usually required for transplantation into patients. We established several cell eMSC lines and cultivated them over a long period to examine the possibility of spontaneous transformation. All cell lines exhibit limited lifespan, undergo replicative senescence, and die. Karyotypic analysis upon different passages reveals that most cells display karyotypic stability. Thus, extended in vitro cultivation of eMSCs does not lead to spontaneous transformation, which makes therapeutic application of these cells safe for patients. During long-term cultivation, eMSCs maintain the expression of surface markers.     

Nucleosomes affect local transformation efficiency

Genetic transformation is a natural process during which foreign DNA enters a cell and integrates into the genome. Apart from its relevance for horizontal gene transfer in nature, transformation is also the cornerstone of today''s recombinant gene technology. Despite its importance, relatively little is known about the factors that determine transformation efficiency. We hypothesize that differences in DNA accessibility associated with nucleosome positioning may affect local transformation efficiency. We investigated the landscape of transformation efficiency at various positions in the Saccharomyces cerevisiae genome and correlated these measurements with nucleosome positioning. We find that transformation efficiency shows a highly significant inverse correlation with relative nucleosome density. This correlation was lost when the nucleosome pattern, but not the underlying sequence was changed. Together, our results demonstrate a novel role for nucleosomes and also allow researchers to predict transformation efficiency of a target region and select spots in the genome that are likely to yield higher transformation efficiency.     

Nucleosomes: regulators of transcription

Histones and nucleosomes are involved in the folding of DNA in the eukaryotic cell. Recent evidence suggests that they are also involved in a multistep process of DNA unfolding and gene regulation.     

Nucleosomes,transcription, and probability

Speaking of current measurements on single ion channel molecules, David Colquhoun wrote in 2006, “Individual molecules behave randomly, so suddenly we had to learn how to deal with stochastic processes.” Here I describe theoretical efforts to understand recent experimental observations on the chromatin structure of single gene molecules, a molecular biologist''s path toward probabilistic theories.     

Nucleosomes Shape DNA Polymorphism and Divergence

An estimated 80% of genomic DNA in eukaryotes is packaged as nucleosomes, which, together with the remaining interstitial linker regions, generate higher order chromatin structures [1]. Nucleosome sequences isolated from diverse organisms exhibit ∼10 bp periodic variations in AA, TT and GC dinucleotide frequencies. These sequence elements generate intrinsically curved DNA and help establish the histone-DNA interface. We investigated an important unanswered question concerning the interplay between chromatin organization and genome evolution: do the DNA sequence preferences inherent to the highly conserved histone core exert detectable natural selection on genomic divergence and polymorphism? To address this hypothesis, we isolated nucleosomal DNA sequences from Drosophila melanogaster embryos and examined the underlying genomic variation within and between species. We found that divergence along the D. melanogaster lineage is periodic across nucleosome regions with base changes following preferred nucleotides, providing new evidence for systematic evolutionary forces in the generation and maintenance of nucleosome-associated dinucleotide periodicities. Further, Single Nucleotide Polymorphism (SNP) frequency spectra show striking periodicities across nucleosomal regions, paralleling divergence patterns. Preferred alleles occur at higher frequencies in natural populations, consistent with a central role for natural selection. These patterns are stronger for nucleosomes in introns than in intergenic regions, suggesting selection is stronger in transcribed regions where nucleosomes undergo more displacement, remodeling and functional modification. In addition, we observe a large-scale (∼180 bp) periodic enrichment of AA/TT dinucleotides associated with nucleosome occupancy, while GC dinucleotide frequency peaks in linker regions. Divergence and polymorphism data also support a role for natural selection in the generation and maintenance of these super-nucleosomal patterns. Our results demonstrate that nucleosome-associated sequence periodicities are under selective pressure, implying that structural interactions between nucleosomes and DNA sequence shape sequence evolution, particularly in introns.