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Open Access Publications from the University of California

Scholarly Works (6 results)

  • Thesis
  • Peer Reviewed
UC San Diego Electronic Theses and Dissertations (2017)

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.

  • 1 supplemental file
Cover page: Histone demethylase LSD1: Connecting developmental signals, chromatin, and cell response
  • Article
  • Peer Reviewed
UC San Diego Previously Published Works (2021)
FOXA pioneer transcription factors (TFs) associate with primed enhancers in endodermal organ precursors. Using a human stem cell model of pancreas differentiation, we here discover that only a subset of pancreatic enhancers is FOXA-primed, whereas the majority is unprimed and engages FOXA upon lineage induction. Primed enhancers are enriched for signal-dependent TF motifs and harbor abundant and strong FOXA motifs. Unprimed enhancers harbor fewer, more degenerate FOXA motifs, and FOXA recruitment to unprimed but not primed enhancers requires pancreatic TFs. Strengthening FOXA motifs at an unprimed enhancer near NKX6.1 renders FOXA recruitment pancreatic TF-independent, induces priming, and broadens the NKX6.1 expression domain. We make analogous observations about FOXA binding during hepatic and lung development. Our findings suggest a dual role for FOXA in endodermal organ development: first, FOXA facilitates signal-dependent lineage initiation via enhancer priming, and second, FOXA enforces organ cell type-specific gene expression via indirect recruitment by lineage-specific TFs.
    Cover page: Sequence logic at enhancers governs a dual mechanism of endodermal organ fate induction by FOXA pioneer factors
    • Article
    • Peer Reviewed
    UC San Diego Previously Published Works (2020)
    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.
      Cover page: LSD1-mediated enhancer silencing attenuates retinoic acid signalling during pancreatic endocrine cell development
      • Article
      • Peer Reviewed
      UC San Diego Previously Published Works (2019)
      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.
        Cover page: A Network of microRNAs Acts to Promote Cell Cycle Exit and Differentiation of Human Pancreatic Endocrine Cells
        • Article
        • Peer Reviewed
        UC San Diego Previously Published Works (2019)
        Genetic variants affecting pancreatic islet enhancers are central to T2D risk, but the gene targets of islet enhancer activity are largely unknown. We generate a high-resolution map of islet chromatin loops using Hi-C assays in three islet samples and use loops to annotate target genes of islet enhancers defined using ATAC-seq and published ChIP-seq data. We identify candidate target genes for thousands of islet enhancers, and find that enhancer looping is correlated with islet-specific gene expression. We fine-map T2D risk variants affecting islet enhancers, and find that candidate target genes of these variants defined using chromatin looping and eQTL mapping are enriched in protein transport and secretion pathways. At IGF2BP2, a fine-mapped T2D variant reduces islet enhancer activity and IGF2BP2 expression, and conditional inactivation of IGF2BP2 in mouse islets impairs glucose-stimulated insulin secretion. Our findings provide a resource for studying islet enhancer function and identifying genes involved in T2D risk.
          Cover page: Pancreatic islet chromatin accessibility and conformation reveals distal enhancer networks of type 2 diabetes risk
          • Article
          • Peer Reviewed
          UC San Diego Previously Published Works (2023)
          Adaptation of the islet β cell insulin-secretory response to changing insulin demand is critical for blood glucose homeostasis, yet the mechanisms underlying this adaptation are unknown. Here, we have shown that nutrient-stimulated histone acetylation plays a key role in adapting insulin secretion through regulation of genes involved in β cell nutrient sensing and metabolism. Nutrient regulation of the epigenome occurred at sites occupied by the chromatin-modifying enzyme lysine-specific demethylase 1 (Lsd1) in islets. β Cell-specific deletion of Lsd1 led to insulin hypersecretion, aberrant expression of nutrient-response genes, and histone hyperacetylation. Islets from mice adapted to chronically increased insulin demand exhibited shared epigenetic and transcriptional changes. Moreover, we found that genetic variants associated with type 2 diabetes were enriched at LSD1-bound sites in human islets, suggesting that interpretation of nutrient signals is genetically determined and clinically relevant. Overall, these studies revealed that adaptive insulin secretion involves Lsd1-mediated coupling of nutrient state to regulation of the islet epigenome.
            Cover page: Nutrient regulation of the islet epigenome controls adaptive insulin secretionCreative Commons 'BY-NC-ND' version 4.0 license
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