An EZ Mark to Miss

Ezh2, the methyltransferase within Polycomb Repressive II Complexes, was thought to be essential for all H3K27me3 marks in embryonic stem cells (ESCs). Recently in Molecular Cell, Shen and colleagues (2008) revealed that EZH2 is dispensable for ESC derivation and self-renewal, and that EZH1 may unexpectedly compensate for its loss

Rescue of Degradation-Prone Mutants of the FK506-Rapamycin Binding (FRB) Protein with Chemical Ligands

We recently reported that certain mutations in the FK506-rapamycin binding (FRB) domain disrupt its stability in vitro and in vivo (Stankunas et al. Mol. Cell , 2003 , 12 , 1615). To determine the precise residues that cause instability, we calculated the folding free energy (Δ G ) of a collection of FRB mutants by measuring their intrinsic tryptophan fluorescence during reversible chaotropic denaturation. Our results implicate the T2098L point mutation as a key determinant of instability. Further, we found that some of the mutants in this collection were destabilised by up to 6 kcal mol −1 relative to the wild type. To investigate how these mutants behave in cells, we expressed firefly luciferase fused to FRB mutants in African green monkey kidney (COS) cell lines and mouse embryonic fibroblasts (MEFs). When unstable FRB mutants were used, we found that the protein levels and the luminescence intensities were low. However, addition of a chemical ligand for FRB, rapamycin, restored luciferase activity. Interestingly, we found a roughly linear relationship between the Δ G of the FRB mutants calculated in vitro and the relative chemical rescue in cells. Because rapamycin is capable of simultaneously binding both FRB and the chaperone, FK506-binding protein (FKBP), we next examined whether FKBP might contribute to the protection of FRB mutants. Using both in vitro experiments and a cell-based model, we found that FKBP stabilizes the mutants. These findings are consistent with recent models that suggest damage to intrinsic Δ G can be corrected by pharmacological chaperones. Further, these results provide a collection of conditionally stable fusion partners for use in controlling protein stability.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/56088/1/1162_ftp.pd

Chd8 Mutation Leads to Autistic-like Behaviors and Impaired Striatal Circuits

Autism spectrum disorder (ASD) is a heterogeneous disease, but genetically defined models can provide an entry point to studying the molecular underpinnings of this disorder. We generated germline mutant mice with loss-of-function mutations in Chd8, a de novo mutation strongly associated with ASD, and demonstrate that these mice display hallmark ASD behaviors, macrocephaly, and craniofacial abnormalities similar to patient phenotypes. Chd8[superscript +/–] mice display a broad, brain-region-specific dysregulation of major regulatory and cellular processes, most notably histone and chromatin modification, mRNA and protein processing, Wnt signaling, and cell-cycle regulation. We also find altered synaptic physiology in medium spiny neurons of the nucleus accumbens. Perturbation of Chd8 in adult mice recapitulates improved acquired motor learning behavior found in Chd8[superscript +/–] animals, suggesting a role for CHD8 in adult striatal circuits. These results support a mechanism linking chromatin modification to striatal dysfunction and the molecular pathology of ASD.National Science Foundation (U.S.) (1122374)National Science Foundation (U.S.) (2013169249)National Institute of Mental Health (U.S.) (F31-MH111157)Howard Hughes Medical Institute (NS046789)Simons Foundation Autism Research Initiative (306063)Simons Foundation Autism Research Initiative (6927482)National Institute of Mental Health (U.S.) (5DP1-MH100706)National Institute of Mental Health (U.S.) (1R01-MH110049)Nancy Lurie Marks Family Foundation (6928117)Howard Hughes Medical Institute (NS046789

Relationships between Remotely Sensed Data and Biomass Components in a Big Sagebrush (Artemisia tridentata) Dominated Area in Yellowstone National Park

Abstract: The predictive power of a hyperspectral imagery for estimating woody and herbaceous biomass were examined for a big sagebrush (Artemisia tridentata) dominated area in Yellowstone National Park, Wyoming, United States of America. The normalized difference vegetation (NDV) and structure insensitive pigment (SIP) indices were used to investigate the relationships between biomass components and reflectance spectra. Ground data were collected in 13 sample plots 1 m 2 in size by clipping all herbaceous vegetation to ground level and stripping green leaves from big sagebrush plants. Strong relationships (R 2 from 0.83 to 0.96) were found between the hyperspectral data and biomass components. The results indicate that fine resolution hyperspectral imagery is capable of estimating various biomass components in big sagebrush dominated areas

Evolution and Organization of the Fibrinogen Locus on Chromosome 4: Gene Duplication Accompanied by Transposition and Inversion

Human fibrinogen cDNA probes for the alpha-, beta-, and gamma-polypeptide chains have been used to isolate the corresponding genes from human genomic libraries. There is a single copy of each gene. Restriction endonuclease analysis of isolated genomic clones and human genomic DNA indicates that the human alpha-, beta-, and gamma-fibrinogen genes are closely linked in a 50-kilobase region of a single human chromosome: the alpha-gene in the middle flanked by the beta-gene on one side and the gamma-gene on the other. The alpha- and gamma-chain genes are oriented in tandem and transcribed toward the beta-chain gene. The beta-chain gene is transcribed from the opposite DNA strand toward the gamma- and alpha-chain genes. The three genes have been localized to the distal third of the long arm of chromosome 4, bands q23-q32, by in situ hybridization with fibrinogen cDNAs and by examination of DNA from multiple rodent-human somatic cell hybrids. Alternative explanations for the present arrangement of the three fibrinogen genes involve either a three-step mechanism with inversion of the alpha /gamma-region or a two-step mechanism involving remote transposition and inversion. The second more simple mechanism has a precedent in the origin of repeated regions of the fibrinogen and immunoglobulin genes

Kinetic analysis of npBAF to nBAF switching reveals exchange of SS18 with CREST and integration with neural developmental pathways

During the development of the vertebrate nervous system, neural progenitors divide, generate progeny that exit mitosis, and then migrate to sites where they elaborate specific morphologies and synaptic connections. Mitotic exit in neurons is accompanied by an essential switch in ATP-dependent chromatin regulatory complexes from the neural progenitor Brg/Brm-associated factor (npBAF) to neuron-specific nBAF complexes that is in part driven by miR-9/9* and miR-124. Recapitulating this microRNA/chromatin switch in fibroblasts leads to their direct conversion to neurons. We have defined the kinetics of neuron-specific BAF complex assembly in the formation of induced neurons from mouse embryonic stem cells, human fibroblasts, and normal mouse neural differentiation and, using proteomic analysis, found that this switch also includes the removal of SS18 and its replacement by CREST at mitotic exit. We found that switching of chromatin remodeling mechanisms is highly correlated with a broad switch in the use of neurogenic transcription factors. Knock-down of SS18 in neural stem cells causes cell-cycle exit and failure to self-renew, whereas continued expression of SS18 in neurons blocks dendritic outgrowth, underlining the importance of subunit switching. Because dominant mutations in BAF subunits underlie widely different human neurologic diseases arising in different neuronal types, our studies suggest that the characteristics of these diseases must be interpreted in the context of the different BAF assemblies in neurons rather than a singular mammalian SWItch/sucrose nonfermentable (mSWI/SNF) complex

Murine esBAF chromatin remodeling complex subunits BAF250a and Brg1 are necessary to maintain and reprogram pluripotency-specific replication timing of select replication domains

Background: Cellular differentiation and reprogramming are accompanied by changes in replication timing and 3D organization of large-scale (400 to 800 Kb) chromosomal domains (‘replication domains’), but few gene products have been identified whose disruption affects these properties. Results: Here we show that deletion of esBAF chromatin-remodeling complex components BAF250a and Brg1, but not BAF53a, disrupts replication timing at specific replication domains. Also, BAF250a-deficient fibroblasts reprogrammed to a pluripotency-like state failed to reprogram replication timing in many of these same domains. About half of the replication domains affected by Brg1 loss were also affected by BAF250a loss, but a much larger set of domains was affected by BAF250a loss. esBAF binding in the affected replication domains was dependent upon BAF250a but, most affected domains did not contain genes whose transcription was affected by loss of esBAF. Conclusions: Loss of specific esBAF complex subunits alters replication timing of select replication domains in pluripotent cells

The Transcription Factor NF-ATc1 Regulates Lymphocyte Proliferation and Th2 Cytokine Production

AbstractNF-ATc1 is a member of a family of genes that encodes the cytoplasmic component of the nuclear factor of activated T cells (NF-AT). In activated T cells, nuclear NF-AT binds to the promoter regions of multiple cytokine genes and induces their transcription. The role of NF-ATc1 was investigated in recombination activating gene-1 (RAG-1)–deficient blastocyst complementation assays using homozygous NF-ATc1−/− mutant ES cell lines. NF-ATc1−/−/RAG-1−/− chimeric mice showed reduced numbers of thymocytes and impaired proliferation of peripheral lymphocytes, but normal production of IL-2. Induction in vitro of Th2 responses, as demonstrated by a decrease in IL-4 and IL-6 production, was impaired in mutant T cells. These data indicate that NF-ATc1 plays roles in the development of T lymphocytes and in the differentiation of the Th2 response

Dynamics of BAF- Polycomb Complex Opposition on Heterochromatin in Normal and Oncogenic States

The opposition between polycomb repressive complexes (PRC) and BAF (mSWI/SNF) complexes plays critical roles in development and disease. Mutations in the genes encoding BAF subunits contribute to over 20% of human malignancy, yet the underlying mechanisms remain unclear owing largely to a lack of assays to assess BAF function in vivo. To address this, we have developed a widely applicable recruitment assay system and find that BAF opposes PRC by rapid, ATP-dependent eviction, leading to the formation of accessible chromatin. Reversing this process results in reassembly of facultative heterochromatin. Surprisingly, BAF-mediated PRC eviction occurs in the absence of PolII occupancy, transcription, and replication. Further, we find that tumor suppressor and oncogenic BAF complex mutations result in differential effects on PRC eviction. These studies define a mechanistic sequence underlying the resolution and formation of facultative heterochromatin and demonstrate that BAF opposes polycomb complexes on a minute-by-minute basis to provide epigenetic plasticity

Addressing Antimicrobial Resistance in China: Policy Implementation in a Complex Context

The effectiveness of antibiotics in treating bacterial infections is decreasing in China because of the widespread development of resistant organisms. Although China has enacted a number of regulations to address this problem, but the impact is very limited. This paper investigates the implementation of these regulations through the lens of complex adaptive systems (CAS). It presents the findings from reviews of relevant policy documents and published papers. The paper identifies different types of agent and explores their interaction with regard to the use of antibiotics and their responses to changes of the regulations. It focuses particularly on the impact of perverse financial incentives on overall patterns of use of antibiotics. Implications for the possibilities of nonlinear results, interactive relationships, and new pathways of policy implementation are discussed. The paper concludes that policy-makers need to better understand the objectives, incentives and potential adaptive behaviors of the agents when they implement interventions to improve antibiotic use and reduce the risk of emergence of resistant organism