DNA methylation directs genomic localization of Mbd2 and Mbd3 in embryonic stem cells

Cytosine methylation is an epigenetic and regulatory mark that functions in part through recruitment of chromatin remodeling complexes containing methyl-CpG binding domain (MBD) proteins. Two MBD proteins, Mbd2 and Mbd3, were previously shown to bind methylated or hydroxymethylated DNA, respectively; however, both of these findings have been disputed. Here, we investigated this controversy using experimental approaches and re-analysis of published data and find no evidence for methylation-independent functions of Mbd2 or Mbd3. We show that chromatin localization of Mbd2 and Mbd3 is highly overlapping and, unexpectedly, we find Mbd2 and Mbd3 are interdependent for chromatin association. Further investigation reveals that both proteins are required for normal levels of cytosine methylation and hydroxymethylation in murine embryonic stem cells. Furthermore, Mbd2 and Mbd3 regulate overlapping sets of genes that are also regulated by DNA methylation/hydroxymethylation factors. These findings reveal an interdependent regulatory mechanism mediated by the DNA methylation machinery and its readers

Rlim-Dependent and -Independent Pathways for X Chromosome Inactivation in Female ESCs

During female mouse embryogenesis, two forms of X chromosome inactivation (XCI) ensure dosage compensation from sex chromosomes. Beginning at the four-cell stage, imprinted XCI (iXCI) exclusively silences the paternal X (Xp), and this pattern is maintained in extraembryonic cell types. Epiblast cells, which give rise to the embryo proper, reactivate the Xp (XCR) and undergo a random form of XCI (rXCI) around implantation. Both iXCI and rXCI depend on the long non-coding RNA Xist. The ubiquitin ligase RLIM is required for iXCI in vivo and occupies a central role in current models of rXCI. Here, we demonstrate the existence of Rlim-dependent and Rlim-independent pathways for rXCI in differentiating female ESCs. Upon uncoupling these pathways, we find more efficient Rlim-independent XCI in ESCs cultured under physiological oxygen conditions. Our results revise current models of rXCI and suggest that caution must be taken when comparing XCI studies in ESCs and mice

Teaching the intangible : how early childhood teacher education instructors "teach" relational development

This thesis set out with the research question, “How are relationships framed, valued, taught and assessed by early childhood educator program instructors in British Columbia?” I conducted six group interviews and five individual interviews with instructors and directors, respectively, at public and private institutions around British Columbia. Using narrative analysis, I constructed a composite instructor character and a composite student instructor character and, using Ollerenshaw and Creswell’s (2002) problem-solution strategy, analyzed the characters during a chronological school year to illustrate tensions that arose at specific points. Overall, instructors frame relationships as foundational in the Early Childhood Educator Program. I draw parallels between the struggle to support adult students while being responsible to children and the balance between pedagogical and andragogical principles. Modeling and engaging in authentic professional relationships with students were the most effective tools for teaching relational development. Instructors engaged in an editing process to ensure that their actions reflected their beliefs, but were still professional. They noted that relational skills can be difficult to assess, and that they cannot assess a student’s willingness to use appropriate skills when needed. In the discussion, I trace the findings back to the purpose and questions for the research. I draw lines between instructors’ discursive constructions of students and Langford’s (2007) Good ECE, and examine the small but distinct cluster of instructors who spoke of the reconceptualising movement and its bearing on a teacher education program.Education, Faculty ofGraduat

Forerunner

Forerunner is a mystery adventure game set in a steampunk science fiction world, developed to explore approaches to dynamic story generation and replayability. Forerunner uses a narrative engine specifically designed for this project that combines dialogue and action in a single story tree

Unbiased chromatin accessibility profiling by RED-seq uncovers unique features of nucleosome variants in vivo

BACKGROUND: Differential accessibility of DNA to nuclear proteins underlies the regulation of numerous cellular processes. Although DNA accessibility is primarily determined by the presence or absence of nucleosomes, differences in nucleosome composition or dynamics may also regulate accessibility. Methods for mapping nucleosome positions and occupancies genome-wide (MNase-seq) have uncovered the nucleosome landscapes of many different cell types and organisms. Conversely, methods specialized for the detection of large nucleosome-free regions of chromatin (DNase-seq, FAIRE-seq) have uncovered numerous gene regulatory elements. However, these methods are less successful in measuring the accessibility of DNA sequences within nucelosome arrays. RESULTS: Here we probe the genome-wide accessibility of multiple cell types in an unbiased manner using restriction endonuclease digestion of chromatin coupled to deep sequencing (RED-seq). Using this method, we identified differences in chromatin accessibility between populations of cells, not only in nucleosome-depleted regions of the genome (e.g., enhancers and promoters), but also within the majority of the genome that is packaged into nucleosome arrays. Furthermore, we identified both large differences in chromatin accessibility in distinct cell lineages and subtle but significant changes during differentiation of mouse embryonic stem cells (ESCs). Most significantly, using RED-seq, we identified differences in accessibility among nucleosomes harboring well-studied histone variants, and show that these differences depend on factors required for their deposition. CONCLUSIONS: Using an unbiased method to probe chromatin accessibility genome-wide, we uncover unique features of chromatin structure that are not observed using more widely-utilized methods. We demonstrate that different types of nucleosomes within mammalian cells exhibit different degrees of accessibility. These findings provide significant insight into the regulation of DNA accessibility

WIDER Working Paper No. 2013/044 Foreign aid and decentralization Policies for autonomy and programming for responsiveness

Donor support for decentralization comes in two main categories: recommendations at the policy level and project activities at the programming level. At the policy level, donors promote decentralization by recommending greater autonomy for subnational actors. That is, they advocate for reforms that increase the extent (or ‘quantity’) of decentralization. At the programming level, donors implement projects intended to improve the capacity and accountability (or ‘quality’) of decentralized governance. This paper’s argument is twofold. First, donors have had modest impacts on the quantity of decentralization where they have engaged in policy reform because the variables that shape the extent of decentralization …