LSD1-mediated enhancer silencing attenuates retinoic acid signalling during pancreatic endocrine cell development.

Developmental progression depends on temporally defined changes in gene expression mediated by transient exposure of lineage intermediates to signals in the progenitor niche. To determine whether cell-intrinsic epigenetic mechanisms contribute to signal-induced transcriptional responses, here we manipulate the signalling environment and activity of the histone demethylase LSD1 during differentiation of hESC-gut tube intermediates into pancreatic endocrine cells. We identify a transient requirement for LSD1 in endocrine cell differentiation spanning a short time-window early in pancreas development, a phenotype we reproduced in mice. Examination of enhancer and transcriptome landscapes revealed that LSD1 silences transiently active retinoic acid (RA)-induced enhancers and their target genes. Furthermore, prolonged RA exposure phenocopies LSD1 inhibition, suggesting that LSD1 regulates endocrine cell differentiation by limiting the duration of RA signalling. Our findings identify LSD1-mediated enhancer silencing as a cell-intrinsic epigenetic feedback mechanism by which the duration of the transcriptional response to a developmental signal is limited

A Network of microRNAs Acts to Promote Cell Cycle Exit and Differentiation of Human Pancreatic Endocrine Cells.

Pancreatic endocrine cell differentiation is orchestrated by the action of transcription factors that operate in a gene regulatory network to activate endocrine lineage genes and repress lineage-inappropriate genes. MicroRNAs (miRNAs) are important modulators of gene expression, yet their role in endocrine cell differentiation has not been systematically explored. Here we characterize miRNA-regulatory networks active in human endocrine cell differentiation by combining small RNA sequencing, miRNA over-expression, and network modeling approaches. Our analysis identified Let-7g, Let-7a, miR-200a, miR-127, and miR-375 as endocrine-enriched miRNAs that drive endocrine cell differentiation-associated gene expression changes. These miRNAs are predicted to target different transcription factors, which converge on genes involved in cell cycle regulation. When expressed in human embryonic stem cell-derived pancreatic progenitors, these miRNAs induce cell cycle exit and promote endocrine cell differentiation. Our study delineates the role of miRNAs in human endocrine cell differentiation and identifies miRNAs that could facilitate endocrine cell reprogramming

Structural and effective connectivity reveals potential network-based influences on category-sensitive visual areas

Visual category perception is thought to depend on brain areas that respond specifically when certain categories are viewed. These category-sensitive areas are often assumed to be modules (with some degree of processing autonomy) and to act predominantly on feedforward visual input. This modular view can be complemented by a view that treats brain areas as elements within more complex networks and as influenced by network properties. This network-oriented viewpoint is emerging from studies using either diffusion tensor imaging to map structural connections or effective connectivity analyses to measure how their functional responses influence each other. This literature motivates several hypotheses that predict category-sensitive activity based on network properties. Large, long-range fiber bundles such as inferior fronto-occipital, arcuate and inferior longitudinal fasciculi are associated with behavioural recognition and could play crucial roles in conveying backward influences on visual cortex from anterior temporal and frontal areas. Such backward influences could support top-down functions such as visual search and emotion-based visual modulation. Within visual cortex itself, areas sensitive to different categories appear well-connected (e.g., face areas connect to object- and motion sensitive areas) and their responses can be predicted by backward modulation. Evidence supporting these propositions remains incomplete and underscores the need for better integration of DTI and functional imaging

Histone demethylase LSD1: Connecting developmental signals, chromatin, and cell response

Over the course of development, regulation of gene transcription is the main mechanism by which pluripotent stem cells become restricted to the various distinct cell types found in the mature organism. Among the many different processes that regulate gene transcription, is the control of physical access to DNA and the genes for which it codes. DNA wound around histone proteins forms chromatin and the enzymes that modify the landscape of that chromatin control which regulatory elements, like promoters and enhancers, are active. This process confers different developmental competencies in cells, enabling them to respond uniquely to similar environmental and developmental signals, regulating gene transcription in turn. The study of these processes during in vitro differentiation of stem cells has enabled us and others to draw links between chromatin remodelers, transcription factors and cellular response to inductive cues during human development.In Chapter 1, I explore the role of the lysine-specific demethylase (LSD1) during human pancreatic development using an in vitro system to differentiate human embryonic stem cells (hESCs) to the pancreatic endocrine lineage. Removal of LSD1 activity during a specific early time window of pancreatic development prevents endocrine formation. Investigation into enhancer regions occupied by LSD1 during this critical time window provided results that support a model in which LSD1-mediated decommissioning renders these enhancers insensitive to activation by external retinoic acid signaling.In Chapter 2, I report my previous work dissecting the role of the transcription factor neurogenin-3 (NGN3) during human pancreatic development. Using the aforementioned hESC-based in vitro differentiation system, gain and loss-of-function studies showed that NGN3 is both necessary and sufficient to induce endocrine formation in human cells.A final supplemental chapter provides an example of a hESC-based pancreatic differentiation protocol similar to the one employed for the studies outlined in Chapters 1 and 2 and discusses the importance of such model systems in dissecting the myriad mechanisms of human disease and development

Preparation and characterization of ultrathin layers of substituted oligo- and poly(p-phenylene)s and mixed layers with octadecanethiol on gold and copper

Substituted poly(p-phenylene)s were adsorbed from solution onto gold and copper and oligo(p-phenylene)s onto gold. The layers were investigated with IR spectroscopy at grazing incidence reflection, XPS, NEXAFS, ToF-SIMS, surface profilometry, AFM, SEM, optical microscopy, ellipsometry, and contact angle measurements to examine their formation and structure. The structure and the properties of the investigated layers depend not only on the chemical structure of the polymer but also on the type of substrate. On gold, the polymers form layers of 15-25 angstroms in thickness and the oligomers of ca. 5 angstroms in thickness. On copper, `thick' layers of up to 900 angstroms were also observed. The oligomers have a lower affinity to gold than the polymers. Mixed octadecanethiol-polymer layers were prepared by immersion of polymer-coated substrates in an octadecanethiol solution or by exposure of self-assembled monolayers of octadecanethiol to polymer solutions. The structure of the mixed layers depends on the sequence of the exposure of the two components and on the chemical structure of the polymer. In the mixed layers, structures that protrude above the surroundings were frequently detected at the surface.</p

A Network of microRNAs Acts to Promote Cell Cycle Exit and Differentiation of Human Pancreatic Endocrine Cells.

Pancreatic endocrine cell differentiation is orchestrated by the action of transcription factors that operate in a gene regulatory network to activate endocrine lineage genes and repress lineage-inappropriate genes. MicroRNAs (miRNAs) are important modulators of gene expression, yet their role in endocrine cell differentiation has not been systematically explored. Here we characterize miRNA-regulatory networks active in human endocrine cell differentiation by combining small RNA sequencing, miRNA over-expression, and network modeling approaches. Our analysis identified Let-7g, Let-7a, miR-200a, miR-127, and miR-375 as endocrine-enriched miRNAs that drive endocrine cell differentiation-associated gene expression changes. These miRNAs are predicted to target different transcription factors, which converge on genes involved in cell cycle regulation. When expressed in human embryonic stem cell-derived pancreatic progenitors, these miRNAs induce cell cycle exit and promote endocrine cell differentiation. Our study delineates the role of miRNAs in human endocrine cell differentiation and identifies miRNAs that could facilitate endocrine cell reprogramming

LSD1-mediated enhancer silencing attenuates retinoic acid signalling during pancreatic endocrine cell development.

Developmental progression depends on temporally defined changes in gene expression mediated by transient exposure of lineage intermediates to signals in the progenitor niche. To determine whether cell-intrinsic epigenetic mechanisms contribute to signal-induced transcriptional responses, here we manipulate the signalling environment and activity of the histone demethylase LSD1 during differentiation of hESC-gut tube intermediates into pancreatic endocrine cells. We identify a transient requirement for LSD1 in endocrine cell differentiation spanning a short time-window early in pancreas development, a phenotype we reproduced in mice. Examination of enhancer and transcriptome landscapes revealed that LSD1 silences transiently active retinoic acid (RA)-induced enhancers and their target genes. Furthermore, prolonged RA exposure phenocopies LSD1 inhibition, suggesting that LSD1 regulates endocrine cell differentiation by limiting the duration of RA signalling. Our findings identify LSD1-mediated enhancer silencing as a cell-intrinsic epigenetic feedback mechanism by which the duration of the transcriptional response to a developmental signal is limited