Browsing by Subject "enhancer"

Now showing 1 - 3 of 3
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    Editing the Neuronal Genome: a CRISPR View of Chromatin Regulation in Neuronal Development, Function, and Plasticity.
    (Yale J Biol Med, 2016-12) Yang, Marty G; West, Anne E
    The dynamic orchestration of gene expression is crucial for the proper differentiation, function, and adaptation of cells. In the brain, transcriptional regulation underlies the incredible diversity of neuronal cell types and contributes to the ability of neurons to adapt their function to the environment. Recently, novel methods for genome and epigenome editing have begun to revolutionize our understanding of gene regulatory mechanisms. In particular, the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has proven to be a particularly accessible and adaptable technique for genome engineering. Here, we review the use of CRISPR/Cas9 in neurobiology and discuss how these studies have advanced understanding of nervous system development and plasticity. We cover four especially salient applications of CRISPR/Cas9: testing the consequences of enhancer mutations, tagging genes and gene products for visualization in live cells, directly activating or repressing enhancers in vivo, and manipulating the epigenome. In each case, we summarize findings from recent studies and discuss evolving adaptations of the method.
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    Epstein-Barr virus ensures B cell survival by uniquely modulating apoptosis at early and late times after infection.
    (Elife, 2017-04-20) Price, Alexander M; Dai, Joanne; Bazot, Quentin; Patel, Luv; Nikitin, Pavel A; Djavadian, Reza; Winter, Peter S; Salinas, Cristina A; Barry, Ashley Perkins; Wood, Kris C; Johannsen, Eric C; Letai, Anthony; Allday, Martin J; Luftig, Micah A
    Latent Epstein-Barr virus (EBV) infection is causally linked to several human cancers. EBV expresses viral oncogenes that promote cell growth and inhibit the apoptotic response to uncontrolled proliferation. The EBV oncoprotein LMP1 constitutively activates NFκB and is critical for survival of EBV-immortalized B cells. However, during early infection EBV induces rapid B cell proliferation with low levels of LMP1 and little apoptosis. Therefore, we sought to define the mechanism of survival in the absence of LMP1/NFκB early after infection. We used BH3 profiling to query mitochondrial regulation of apoptosis and defined a transition from uninfected B cells (BCL-2) to early-infected (MCL-1/BCL-2) and immortalized cells (BFL-1). This dynamic change in B cell survival mechanisms is unique to virus-infected cells and relies on regulation of MCL-1 mitochondrial localization and BFL-1 transcription by the viral EBNA3A protein. This study defines a new role for EBNA3A in the suppression of apoptosis with implications for EBV lymphomagenesis.
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    The Glucocorticoid-Mediated Dynamics of Genome Architecture
    (2018) D'Ippolito, Anthony

    Human cells are perpetually receiving and responding to a variety of intrinsic and extrinsic signals. A primary mechanism by which cells carry out these responses is via changes in the regulation of gene expression. Many studies have examined gene regulation in steady state systems, but few have investigated the genomic response to stimuli. Therefore, it is less well understood how cellular stimuli elicit dynamic gene expression responses. Here, we investigate how extracellular stimuli mediate gene expression responses via: 1) Changes in transcription factor configurations at enhancer elements; and 2) Changes in chromatin looping between putative enhancers and their target gene promoters. To study these phenomena, we used glucocorticoid (GC) treatment as a model transcriptional stimulus. This hormone steroid is known to bind to and activate the GC receptor (GR), a ligand-induced transcription factor (TF), and is therefore a highly tractable system for studying stimulus responsive gene regulation. Using this model system, we first used high-resolution TF-binding site mapping approaches to elucidate the genomic binding locations of GR and its associated cofactors. Using these approaches, we found evidence that: 1) The GR binds to the genome as both a monomer and dimer; and 2) The GR binds to the genome with AP-1 in a more relaxed configuration, while it binds FOXA1 in a more constrained configuration. We next interrogated the role of chromatin looping in mediating dynamic transcriptional responses. For this work we used high-throughput genomics methods to assay chromatin conformation across a time course of GC treatment. These studies resulted in several main findings: 1) Chromatin loops do not form in response to stimulus, but are instead pre-formed before GC treatment; 2) Chromatin looping interactions increase between distal GR binding sites and GC-responsive genes; 3) The insulator protein CTCF is depleted at stimulus responsive looping interactions; and 4) GC treatment mediates changes in higher-order chromosome compartmentalization that are concordant with gene expression responses. Together these results provide evidence for a genome topology that is pre-wired to respond to stimulus, and that subsequent transcriptional responses are mediated through GR binding to putative enhancer elements with other TFs, in a variety of genomic binding configurations.