Three Dimensional Dual Labelled DNA Fluorescent In Situ Hybridization Analysis in Fixed Tissue Sections

Emerging studies demonstrate that three-dimensional organization of chromatin in the nucleus plays a vital role in regulating the genome. DNA fluorescent in situ hybridization (FISH) is a common molecular technique used to visualize the location of DNA sequences. The vast majority of DNA FISH studies are conducted on cultured cells due to the technical difficulties encountered using fixed tissue sections. However, the use of cultured cells poses important limitations that could yield misleading results, making in vivo analysis a far superior approach. Here we present a protocol for multiplexed three dimensional DNA FISH in mouse brain sections, which is also applicable to other tissues. Paraffin-embedded tissues could be used but the embedding and preparation of the samples is time-consuming and often associated with poor antigenicity. To overcome this problem we:•developed a FISH technique using fixed, frozen cryosections;•provide specific instructions for tissue processing for proper fixation and freezing, including equilibration in sucrose gradients to maintain proper cellular structure;•include optimized permeabilization and washing steps to achieve specific signal and to limit background fluorescence in tissue sections

ATRX promotes gene expression by facilitating transcriptional elongation through guanine-rich coding regions

ATRX is a chromatin remodeling protein involved in deposition of the histone variant H3.3 at telomeres and pericentromeric heterochromatin. It also influences the expression level of specific genes; however, deposition of H3.3 at transcribed genes is currently thought to occur independently of ATRX. We focused on a set of genes, including the autism susceptibility gene Neuroligin 4 (Nlgn4), that exhibit decreased expression in ATRX-null cells to investigate the mechanisms used by ATRX to promote gene transcription. Overall TERRA levels, as well as DNA methylation and histone modifications at ATRX target genes are not altered and thus cannot explain transcriptional dysregulation.We found thatATRX does not associate with the promoter of these genes, but rather binds within regions of the gene body corresponding to high H3.3 occupancy. These intragenic regions consist of guanine-rich DNA sequences predicted to form non-B DNA structures called G-quadruplexes during transcriptional elongation.We demonstrate thatATRX deficiency corresponds to reduced H3.3 incorporation and stalling ofRNApolymerase II at these G-rich intragenic sites. These findings suggest that ATRX promotes the incorporation of histone H3.3 at particular transcribed genes and facilitates transcriptional elongation through G-rich sequences. The inability to transcribe genes such as Nlgn4 could cause deficits in neuronal connectivity and cognition associated with ATRX mutations in humans

The SWI/SNF protein ATRX co-regulates pseudoautosomal genes that have translocated to autosomes in the mouse genome

<p>Abstract</p> <p>Background</p> <p>Pseudoautosomal regions (PAR1 and PAR2) in eutherians retain homologous regions between the X and Y chromosomes that play a critical role in the obligatory X-Y crossover during male meiosis. Genes that reside in the PAR1 are exceptional in that they are rich in repetitive sequences and undergo a very high rate of recombination. Remarkably, murine PAR1 homologs have translocated to various autosomes, reflecting the complex recombination history during the evolution of the mammalian X chromosome.</p> <p>Results</p> <p>We now report that the SNF2-type chromatin remodeling protein ATRX controls the expression of eutherian ancestral PAR1 genes that have translocated to autosomes in the mouse. In addition, we have identified two potentially novel mouse PAR1 orthologs.</p> <p>Conclusion</p> <p>We propose that the ancestral PAR1 genes share a common epigenetic environment that allows ATRX to control their expression.</p

Dual effect of CTCF loss on neuroprogenitor differentiation and survival

An increasing number of proteins involved in genome organization have been implicated in neurodevelopmental disorders, highlighting the importance of chromatin architecture in the developing CNS. The CCCTC-binding factor (CTCF) is a zinc finger DNA binding protein involved in higher-order chromatin organization, and mutations in the human CTCF gene cause an intellectual disability syndrome associated with microcephaly. However, information on CTCF function in vivo in the developing brain is lacking. To address this gap, we conditionally inactivated the Ctcf gene at early stages of mouse brain development. Cre-mediated Ctcf deletion in the telencephalon and anterior retina at embryonic day 8.5 triggered upregulation of the p53 effector PUMA (p53 upregulated modulator of apoptosis), resulting in massive apoptosis and profound ablation of telencephalic structures. Inactivation of Ctcf several days later at E11 also resulted in PUMA upregulation and increased apoptotic cell death, and the Ctcf-null forebrain was hypocellular and disorganized at birth. Although deletion of both Ctcf and Puma in the embryonic brain efficiently rescued Ctcf-null progenitor cell apoptosis, it failed to improve neonatal hypocellularity due to decreased proliferative capacity of rescued apical and outer radial glia progenitor cells. This was exacerbated by an independent effect of CTCF loss that resulted in depletion of the progenitor pool due to premature neurogenesis earlier in development. Our findings demonstrate that CTCF activities are required for two distinct events in early cortex formation: first, to correctly regulate the balance between neuroprogenitor cell proliferation and differentiation, and second, for the survival of neuroprogenitor cells, providing new clues regarding the contributions of CTCF in microcephaly/intellectual disability syndrome pathologies. © 2014 the authors

Thermodynamic properties of metal hydride nanostructures

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Physics, February 2009."September 2008." Cataloged from PDF version of thesis.Includes bibliographical references (p. 235-243).Hydrogen is considered a good energy carrier candidate for future automotive applications because of its high abundance and its potential role in a carbon-free cycle. The high gravimetric and volumetric storage capacities of metal hydrides make them ideal hydrogen carriers if the limitations associated with their slow hydrogen release kinetics, their high hydrogen release temperatures, and their poor thermal properties can be resolved. In this thesis, the thermodynamic and kinetic improvements on the hydrogen release properties of nanostructured metal hydrides are investigated both theoretically and experimentally. Four main results are presented in this work. The excess volume present in deformed regions is identified as the key factor in explaining the reduction in the enthalpy of formation observed experimentally in nanostructured materials. The impact of excess volume on the enthalpy of formation at OK is quantified using three equations of state, and it is found to be as important as the combined impact of surfaces, grain boundaries and the presence of metastable crystalline phases. Then, the findings on the properties of excess volume are generalized to high temperatures. It is demonstrated that the impact of temperature will be more favorable to a reduction of the enthalpy of formation if a large fraction of the metal hydride is in a state of small excess volume compared to a small fraction of the hydride in a state of high excess volume. The impact of a temperature increase on the enthalpy of formation of metal hydrides is found to offset the effect of the excess volume as calculated at OK.(cont.) The stability of the regions containing excess volume over multiple hydriding/dehydriding cycles is also calculated. At high temperatures and large excess volumes, the free energy barrier created by the excess entropy reduces the recrystallization rate of the deformed regions by several orders of magnitude. A regime where the benefits of the excess volume on the enthalpy of formation can be maintained is thus identified. Finally, an experiment to study the cycling properties of metal hydrides was designed using Raman spectroscopy. It is demonstrated that the release temperature of hydrogen could be accurately measured using Raman spectroscopy.by Vincent Bérubé.Ph.D

Analysis of neonatal brain lacking ATRX or MeCP2 reveals changes in nucleosome density, CTCF binding and chromatin looping

ATRX and MeCP2 belong to an expanding group of chromatin-associated proteins implicated in human neurodevelopmental disorders, although their gene-regulatory activities are not fully resolved. Loss of ATRX prevents full repression of an imprinted gene network in the postnatal brain and in this study we address the mechanistic aspects of this regulation. We show that ATRX binds many imprinted domains individually but that transient co-localization between imprinted domains in the nuclei of neurons does not require ATRX. We demonstrate that MeCP2 is required for ATRX recruitment and that deficiency of either ATRX or MeCP2 causes decreased frequency of long-range chromatin interactions associated with altered nucleosome density at CTCF-binding sites and reduced CTCF occupancy. These findings indicate that MeCP2 and ATRX regulate gene expression at a subset of imprinted domains by maintaining a nucleosome configuration conducive to CTCF binding and to the maintenance of higher order chromatin structure

Invariant vector fields and the prolongation method for supersymmetric quantum systems

The kinematical and dynamical symmetries of equations describing the time evolution of quantum systems like the supersymmetric harmonic oscillator in one space dimension and the interaction of a non-relativistic spin one-half particle in a constant magnetic field are reviewed from the point of view of the vector field prolongation method. Generators of supersymmetries are then introduced so that we get Lie superalgebras of symmetries and supersymmetries. This approach does not require the introduction of Grassmann valued differential equations but a specific matrix realization and the concept of dynamical symmetry. The Jaynes-Cummings model and supersymmetric generalizations are then studied. We show how it is closely related to the preceding models. Lie algebras of symmetries and supersymmetries are also obtained.Comment: 37 pages, 7 table