Campus Environment 2008: A National Report Card on Sustainability in Higher Education

Presents survey findings on national and regional trends among colleges in environmental leadership in management, academic courses in sustainability, and conservation efforts in operations. Profiles exemplary programs and notes areas for improvement

Setdb1-mediated H3K9 methylation is enriched on the inactive X and plays a role in its epigenetic silencing

Background: The presence of histone 3 lysine 9 (H3K9) methylation on the mouse inactive X chromosome has been controversial over the last 15 years, and the functional role of H3K9 methylation in X chromosome inactivation in any species has remained largely unexplored. Results: Here we report the first genomic analysis of H3K9 di- and tri-methylation on the inactive X: we find they are enriched at the intergenic, gene poor regions of the inactive X, interspersed between H3K27 tri-methylation domains found in the gene dense regions. Although H3K9 methylation is predominantly non-genic, we find that depletion of H3K9 methylation via depletion of H3K9 methyltransferase Set domain bifurcated 1 (Setdb1) during the establishment of X inactivation, results in failure of silencing for around 150 genes on the inactive X. By contrast, we find a very minor role for Setdb1-mediated H3K9 methylation once X inactivation is fully established. In addition to failed gene silencing, we observed a specific failure to silence X-linked long-terminal repeat class repetitive elements. Conclusions: Here we have shown that H3K9 methylation clearly marks the murine inactive X chromosome. The role of this mark is most apparent during the establishment phase of gene silencing, with a more muted effect on maintenance of the silent state. Based on our data, we hypothesise that Setdb1-mediated H3K9 methylation plays a role in epigenetic silencing of the inactive X via silencing of the repeats, which itself facilitates gene silencing through alterations to the conformation of the whole inactive X chromosome.Andrew Keniry, Linden J. Gearing, Natasha Jansz, Joy Liu, Aliaksei Z. Holik, Peter F. Hickey, Sarah A. Kinkel, Darcy L. Moore, Kelsey Breslin, Kelan Chen, Ruijie Liu, Catherine Phillips, Miha Pakusch, Christine Biben, Julie M. Sheridan, Benjamin T. Kile, Catherine Carmichael, Matthew E. Ritchie, Douglas J. Hilton and Marnie E. Blewit

Protein–protein HADDocking using exclusively pseudocontact shifts

In order to enhance the structure determination process of macromolecular assemblies by NMR, we have implemented long-range pseudocontact shift (PCS) restraints into the data-driven protein docking package HADDOCK. We demonstrate the efficiency of the method on a synthetic, yet realistic case based on the lanthanide-labeled N-terminal ε domain of the E. coli DNA polymerase III (ε186) in complex with the HOT domain. Docking from the bound form of the two partners is swiftly executed (interface RMSDs < 1 Å) even with addition of very large amount of noise, while the conformational changes of the free form still present some challenges (interface RMSDs in a 3.1–3.9 Å range for the ten lowest energy complexes). Finally, using exclusively PCS as experimental information, we determine the structure of ε186 in complex with the HOT-homologue θ subunit of the E. coli DNA polymerase III

The quadruplex r(CGG)n destabilizing cationic porphyrin TMPyP4 cooperates with hnRNPs to increase the translation efficiency of fragile X premutation mRNA

The 5′ untranslated region of the FMR1 gene which normally includes 4–55 d(CGG) repeats expands to > 55–200 repeats in carriers of fragile X syndrome premutation. Although the levels of premutation FMR1 mRNA in carrier cells are 5–10-fold higher than normal, the amount of the product FMR protein is unchanged or reduced. We demonstrated previously that premutation r(CGG)n tracts formed quadruplex structures that impeded translation and lowered the efficiency of protein synthesis. Normal translation could be restored in vivo by the quadruplex r(CGG)n destabilizing action of CBF-A and hnRNP A2 proteins. Here we report that the quadruplex-interacting cationic porphyrin TMPyP4 by itself and in cooperation with CBF-A or hnRNP A2 also unfolded quadruplex r(CGG)n and increased the efficiency of translation of 5′-(CGG)99 containing reporter firefly (FL) mRNA. TMPyP4 destabilized in vitro a (CGG)33 intramolecular quadruplex structure and enhanced the translation of 5′-(CGG)99-FL mRNA in a rabbit reticulocyte lysate and in HEK293 cells. The efficiency of translation of (CGG)99-FL mRNA was additively increased in cells exposed to TMPyP4 together with CBF-A. Whereas low doses of TMPyP4, CBF-A or hnRNP A2 by themselves did not affect the in vivo utilization of (CGG)99-FL mRNA, introduction of TMPyP4 together with either protein synergistically augmented its translation efficiency

Unique properties of a subset of human pluripotent stem cells with high capacity for self-renewal.

Archetypal human pluripotent stem cells (hPSC) are widely considered to be equivalent in developmental status to mouse epiblast stem cells, which correspond to pluripotent cells at a late post-implantation stage of embryogenesis. Heterogeneity within hPSC cultures complicates this interspecies comparison. Here we show that a subpopulation of archetypal hPSC enriched for high self-renewal capacity (ESR) has distinct properties relative to the bulk of the population, including a cell cycle with a very low G1 fraction and a metabolomic profile that reflects a combination of oxidative phosphorylation and glycolysis. ESR cells are pluripotent and capable of differentiation into primordial germ cell-like cells. Global DNA methylation levels in the ESR subpopulation are lower than those in mouse epiblast stem cells. Chromatin accessibility analysis revealed a unique set of open chromatin sites in ESR cells. RNA-seq at the subpopulation and single cell levels shows that, unlike mouse epiblast stem cells, the ESR subset of hPSC displays no lineage priming, and that it can be clearly distinguished from gastrulating and extraembryonic cell populations in the primate embryo. ESR hPSC correspond to an earlier stage of post-implantation development than mouse epiblast stem cells

BAF complex-mediated chromatin relaxation is required for establishment of X chromosome inactivation

The process of epigenetic silencing, while fundamentally important, is not yet completely understood. Here we report a replenishable female mouse embryonic stem cell (mESC) system, Xmas, that allows rapid assessment of X chromosome inactivation (XCI), the epigenetic silencing mechanism of one of the two X chromosomes that enables dosage compensation in female mammals. Through a targeted genetic screen in differentiating Xmas mESCs, we reveal that the BAF complex is required to create nucleosome-depleted regions at promoters on the inactive X chromosome during the earliest stages of establishment of XCI. Without this action gene silencing fails. Xmas mESCs provide a tractable model for screen-based approaches that enable the discovery of unknown facets of the female-specific process of XCI and epigenetic silencing more broadly.Andrew Keniry ... Jose M. Polo ... et al

The Formation and Stabilization of a Novel G-Quadruplex in the 5′-Flanking Region of the Relaxin Gene

It has been reported that binding of STAT3 protein to the 5′-flanking region of the relaxin gene may result in downregulation of the relaxin expression. There is a Guanine(G)-rich segment located in about 3.8 Kb upstream of the relaxin gene and very close to the STAT3's binding site. In our study, NMR spectroscopy revealed the formation of G-quadruplex by this G-rich strand, and the result was confirmed by ESI mass spectrometry and CD spectroscopy. The theoretical structure of RLX G-quadruplex was constructed and refined by molecular modeling. When this relaxin G-quadruplex was stabilized by berberine(ΔTm = 10°C), a natural alkaloid from a Chinese herb, the gene expression could be up-regulated in a dose-dependent manner which was proved by luciferase assay. This result is different from the general G-quadruplex function that inhibiting the telomere replication or down-regulating many oncogenes expression. Therefore, our study reported a novel G-quadruplex in the relaxin gene and complemented the regulation mechanism about gene expression by G-quadruplexes

Rare Copy Number Variants Observed in Hereditary Breast Cancer Cases Disrupt Genes in Estrogen Signaling and TP53 Tumor Suppression Network

Breast cancer is the most common cancer in women in developed countries, and the contribution of genetic susceptibility to breast cancer development has been well-recognized. However, a great proportion of these hereditary predisposing factors still remain unidentified. To examine the contribution of rare copy number variants (CNVs) in breast cancer predisposition, high-resolution genome-wide scans were performed on genomic DNA of 103 BRCA1, BRCA2, and PALB2 mutation negative familial breast cancer cases and 128 geographically matched healthy female controls; for replication an independent cohort of 75 similarly mutation negative young breast cancer patients was used. All observed rare variants were confirmed by independent methods. The studied breast cancer cases showed a consistent increase in the frequency of rare CNVs when compared to controls. Furthermore, the biological networks of the disrupted genes differed between the two groups. In familial cases the observed mutations disrupted genes, which were significantly overrepresented in cellular functions related to maintenance of genomic integrity, including DNA double-strand break repair (P = 0.0211). Biological network analysis in the two independent breast cancer cohorts showed that the disrupted genes were closely related to estrogen signaling and TP53 centered tumor suppressor network. These results suggest that rare CNVs represent an alternative source of genetic variation influencing hereditary risk for breast cancer

Psip1/p52 regulates posterior Hoxa genes through activation of lncRNA Hottip

Long noncoding RNAs (lncRNAs) have been implicated in various biological functions including the regulation of gene expression, however, the functionality of lncRNAs is not clearly understood and conflicting conclusions have often been reached when comparing different methods to investigate them. Moreover, little is known about the upstream regulation of lncRNAs. Here we show that the short isoform (p52) of a transcriptional co-activator—PC4 and SF2 interacting protein (Psip1), which is known to be involved in linking transcription to RNA processing, specifically regulates the expression of the lncRNA Hottip–located at the 5’ end of the Hoxa locus. Using both knockdown and knockout approaches we show that Hottip expression is required for activation of the 5’ Hoxa genes (Hoxa13 and Hoxa10/11) and for retaining Mll1 at the 5’ end of Hoxa. Moreover, we demonstrate that artificially inducing Hottip expression is sufficient to activate the 5’ Hoxa genes and that Hottip RNA binds to the 5’ end of Hoxa. By engineering premature transcription termination, we show that it is the Hottip lncRNA molecule itself, not just Hottip transcription that is required to maintains active expression of posterior Hox genes. Our data show a direct role for a lncRNA molecule in regulating the expression of developmentally-regulated mRNA genes in cis