Browsing by Subject "RNA, Long Noncoding"
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Item Epigenetic Regulation of Oligodendrocyte Development and Regeneration in the Central Nervous System(2016-10-26) He, Danyang; Kraus, W. Lee; Lu, Q. Richard; Johnson, Jane E.; Olson, Eric N.Oligodendrocytes (OLs) produce myelin sheaths that electrically insulate axons and promote rapid propagation of action potentials in the CNS. The onset and timing of CNS myelination and remyelination requires precise coordination between epigenetic programming and transcriptional regulation. In this thesis, I present my findings on two epigenetic regulatory complexes Chd7/Sox10 and lncOL1/Suz12 in CNS myelination and remyelination. First, we show that chromatin remodeler Chd7 is required for proper onset of CNS myelination and remyelination. Genome-occupancy analyses, coupled with transcriptome profiling, reveal that Chd7 interacts with Sox10 and targets the enhancers of key myelinogenic genes, and identify novel Chd7 targets including bone formation regulators Osterix/Sp7 and Creb3l2, which are also critical for oligodendrocyte maturation. Thus, Chd7 coordinates with Sox10 to regulate the initiation of myelinogenesis and acts as a molecular nexus of regulatory networks that account for the development of a seemingly diverse array of lineages including oligodendrocytes and osteoblasts, pointing to the hitherto previously uncharacterized Chd7 functions in white matter pathogenesis in CHARGE syndrome. To understand the role of lncRNAs in CNS myelination, we establish dynamic expression profiles of lncRNAs at different stages of oligodendrocyte development and uncover a cohort of stage-specific oligodendrocyte-restricted lncRNAs including a conserved chromatinassociated lncOL1. Genetic inactivation of lncOL1 causes defects in CNS myelination and remyelination following injury. Functional analyses illustrate that lncOL1 interacts with Suz12, a component of PRC2, to promote oligodendrocyte maturation in part through Suz12-mediated repression of a differentiation inhibitory network that maintains the precursor state. Collectively, these studies show that epigenetic circuitry between lncRNAs and transcription factors with chromatin-modifying complexes play roles in balancing inhibitory and activating gene program, allowing the timely CNS myelination and myelin repair.Item Regulation of Pumilio RNA Binding Proteins by Long Noncoding RNA NORAD(August 2021) Elguindy, Mahmoud Mohamed; Zhu, Hao; Buszczak, Michael; Sabari, Benjamin; Mendell, Joshua T.The mammalian genome is extensively transcribed and encodes thousands of long noncoding RNAs (lncRNAs). Defining the mechanism of action of lncRNAs has been a critical challenge and priority for understanding their biological functions. An important example of this is the lncRNA NORAD, which is a highly conserved lncRNA that is required for maintaining genome stability in mammals. Work in this thesis begins by examining the physiologic function and molecular mechanism of NORAD in human cells. We clearly demonstrate that NORAD localizes predominantly to the cytoplasm where it functions by binding to and inhibiting PUMILIO (PUM1 and PUM2) RNA binding proteins (RBPs). Thorough dissection of the functional domains of NORAD establish the essentiality of PUM binding to NORAD for its function in maintaining genome stability. We further examine the mechanism by which NORAD is able to efficiently sequester and regulate PUM proteins. Through a multidisciplinary approach involving biochemical, microscopy, and mutational analysis experiments, we uncover how NORAD sequesters a super-stoichiometric amount of PUM proteins into novel liquid-like membraneless organelles through a multivalency-induced phase separation mechanism. Moreover, we provide a molecular understanding of the properties of NORAD that promote PUM phase separation, revealing new principles of lncRNA function and demonstrating the physiologic importance of RNA-driven phase separation as a regulatory mechanism in mammalian biology. Our findings, together with the widespread repetitive architecture of lncRNAs, suggest that the phase separation principles we establish may be broadly utilized for lncRNA-mediated regulation.Item A Role for POU3F3 in Myocyte Differentiation: Exploring New Frontier in Alveolar Rhabdomyosarcoma Development(2015-01-26) Denegre, Amelia; Granados, Valerie; Avirneni, Usha; Galindo, ReneSUMMARY: Children who are diagnosed with pediatric rhabdomyosarcoma (RMS), a mesenchymal-derived soft tissue cancer that comprises 3.5% of childhood cancers, are often delivered a bleak prognosis with little hope of a future. Despite significant advances illuminating transcription factor signaling in RMS onset and progression, research is still needed to precisely understand RMS pathogenesis on a molecular level in order to develop targeted treatment options. OBJECTIVE: The goal of this project is to explore the role of POU3F3 in myogenesis, particularly in relation to cell fusion and myocyte differentiation. METHODS: Immunofluorescence: POU3F3 knockdown cells were differentiated in 2% horse serum. On day 2 of differentiation, cells were probed with POU3F3 primary and red immunofluorescent secondary antibody, allowing for imaging of POU3F3 localization during myoblast differentiation. Western Blot: Three knock-down shPOU3F3 C2C12 cell-line constructs were tested. Western blots were performed that compared C2C12 control, POU3F3 overexpression, and POU3F3 knockdown cells. Crystal Violet: After differentiation, POU3F3 knock-down and control cell lines were stained with crystal violet stain to visualize the effect of POU3F3 knockdown on differentiation. RESULTS: Immunofluorescence: We confirmed that in knockdown C2C12 cells, POU3F3 localizes like control C2C12, in the periphery. Previous research has shown that in overexpressed POU3F3 C2C12 cell lines, POU3F3 localizes to the nucleus. The implication of the disconnect between POU3F3 location in controls and knock-downs versus overexpressed cell lines is an area that is an opportunity for further research. Western Blot: Western Blot analysis confirmed that POU3F3 knockdown was successful, and provides a platform for further POU3F3 interrogation. Crystal Violet: Crystal violet staining suggests that POU3F3 participates in a myoblast differentiation, as the control cells fuse into myotubes, while POU3F3-silenced cells do not. CONCLUSION: These initial results suggest that POU3F3 participates in muscle differentiation. Next, the Galindo lab will be probing POU3F3 function in myogenesis in greater depth, insights they will next apply to RMS.Item Transcription of the Long Noncoding RNA, UpperHand, Is Required for Heart Development(2016-10-03) Anderson, Kelly Marie; Mendell, Joshua T.; Olson, Eric N.; Graff, Jonathan M.; Conrad, Nicholas; Stull, JamesThe basic helix-loop-helix transcription factor HAND2 is an ancestral regulator of heart development and one of four cardiac transcription factors that controls the reprogramming of fibroblasts to cardiomyocytes. Genetic deletion of Hand2 in mice results in hypoplasia of the right ventricle and embryonic lethality. The embryonic expression of Hand2 is tightly regulated by well-characterized upstream enhancer elements, which reside within a super-enhancer delineated by H3K27ac histone modifications. Here, I show that transcription of a long noncoding RNA upstream of Hand2, which I named UpperHand (UPH), is required to maintain the H3K27ac super-enhancer signature and elongation of RNA polymerase II through the Hand2 locus. Blockade of UPH transcription by insertion of a transcription termination cassette, but not knockdown of the mature transcript, abolished Hand2 expression, causing right ventricular hypoplasia and embryonic lethality in mice. Given the substantial number of uncharacterized promoter-associated lncRNAs encoded by the mammalian genome, the UPH-Hand2 regulatory partnership offers a mechanism by which noncoding transcription can establish a permissive chromatin environment necessary for the expression of a neighboring protein-coding gene.Item Transcriptional Mechanisms Underlying Neuronal Activity-Dependent Plasticity(2016-11-21) Schaukowitch, Katie Marie; Huber, Kimberly M.; Johnson, Jane E.; Tamminga, Carol; Kraus, W. LeeImpairment in learning and memory is a well-established cognitive symptom that is manifested in many psychiatric diseases including autism and schizophrenia. Studies have shown that long-lasting memory formation is mediated by rapid changes in nuclear gene expression in response to learning-induced sensory experience. Despite these findings, there is a significant gap in our knowledge as to how sensory information is precisely translated into specific transcriptional outputs. Recently, a class of long noncoding RNAs that are transcribed bidirectionally from the enhancers of activity-dependent genes in neurons (eRNAs) has been identified. My first project studied the function of eRNAs of two immediate early genes Activity-regulated cytoskeletal protein (Arc) and Growth arrest and DNA-damage-inducible, beta (Gadd45b), which have been implicated in mediating synaptic plasticity. Using a knockdown approach, we found that eRNAs are necessary for the full induction of their target genes in response to membrane depolarization. eRNAs specifically regulate the early elongation stage of transcription by allowing for efficient release of paused RNA polymerase II (RNAPII) from the promoters of activity-regulated genes. Knockdown of eRNAs results in the retention of an RNAPII pausing factor, Negative Elongation Factor (NELF), at the target gene promoter. eRNAs directly bind to NELF during stimulated conditions, suggesting that eRNAs interact with NELF to facilitate its release from the promoter, thus resulting in efficient and precisely timed gene activation. These data define a new role for the spatiotemporally controlled expression of regulatory RNAs in the experience-dependent gene expression network. My second project aimed to identify the transcriptional program activated when activity levels are suppressed. Homeostatic scaling allows neurons to maintain stable activity patterns by globally altering their synaptic strength in response to changing activity levels. Decreasing activity leads to an upregulation in synaptic strength, as seen by increases in AMPA mediated mEPSCs. It was previously shown that the increase in mEPSC amplitude could be blocked by a transcription inhibitor, suggesting that transcription is necessary for the scaling response. However, little is known about the genes directly regulated by activity suppression or the signaling mechanisms underlying the transcriptional control. Using RNA-Seq, we identified nearly 100 genes that were specifically upregulated in response to activity suppression. Neuronal pentraxin-1 (Nptx1), previously shown to promote AMPAR clustering, was increased ~3 fold, and knockdown of this gene blocked the increase in mEPSC amplitudes. SRF is a key transcription factor in regulating Nptx1 induction, which is calcium-dependent, indicating the existence of an active pathway to control transcription. Taken together, this study defines a novel transcriptional program that is able to sense the absence of activity and coordinate the global increase in synaptic strength.