Transcriptome analysis has demonstrated that 70%-90% of the genome is transcribed, yet surprisingly only 1- 2% codes for protein. Recent work has unveiled a new class of non-coding transcript, termed long noncoding RNAs (lncRNAs). A known major function of these lncRNAs is to specifically direct chromatin-modifiers to their targets genome-wide. While bioinformatic advances have lead to extensive cataloguing and functional analysis of lncRNAs in cultured cells, in vivo data is severely lacking. The sub-ventricular zone (SVZ) of the mouse brain represents an ideal system in which to study the role of lncRNAs in vivo. The SVZ contains multipotent neural stem cells (SVZ-NSCs) capable of giving rise to local glial cells as well as terminally differentiated neurons for the olfactory bul (OB). This fate restriction, of stem cells to terminally differentiated cells of the neural lineage is known to be dependent on chromatin-state changes and the actions of key transcription factors. The neural lineage in the SVZ can therefore be used to interrogate the interplay of chromatin state, transcriptional programs, and lncRNAs. The major goal of this work is to characterize the expression and function of a neurogenic lncRNA in vivo. To this end, we have performed RNA-seq and transcriptome reconstruction on RNA isolated from microdissected SVZ and OB tissue. This provided a comprehensive annotation of all lncRNAs expressed in the SVZ and OB, and identified those transcripts specifically enriched in neurogenic brain regions. We combined this RNA-seq analysis with ChIP-seq analysis to identify lncRNA loci that undergo changes in chromatin marks in embryonic stem cells, SVZ-NSCs, and non-neurogenic fibroblasts. This work led us to a transcript annotated as Dlx1as, a 2.8 kb noncoding RNA transcribed from an ultraconserved region adjacent to the Dlx2 gene. Subsequent knockdown experiments in cultured SVZ-NSCs revealed Dlx1as is necessary for efficient neurogenesis in these cultures.
Aim 1 will evaluate whether Dlx1as is necessary to promote neurogenesis in vivo. To test if Dlx1as is necessary, we will inject a knockdown construct into the ventricle of adult mice, and assess the ability of the SVZ-NSCs to produce neurons.
In Aim 2, we will assess whether Dlx1as can direct chromatin modifiers and modifications. Preliminary data reveals that trithorax chromatin modification complex member MLL1 is localized to the Dlx1/2 locus during differentiation, and this localization is RNA-dependent. Is Dlx1as responsible for targeting MLL1 to the Dlx1/2 promoters and/or enhancers? We will use ChIP-qPCR and RNA immunopreciptation (RIP) analysis to assess the chromatin-state changes at the Dlx1/2 locus in Dlx1as-depleted SVZ-NSC cultures. We will determine whether Dlx1as interacts with and potentially recruits specific chromatin remodeling factors through RIP-qPCR. I hypothesize that Dlx1as is required for neurogenesis in vivo through modulation of the chromatin- modifying factors recruited to the Dlx1/2 locus.

Public Health Relevance

Recent advances in sequencing technology have unveiled a new class of RNAs that are abundantly expressed but do not code for protein, called long noncoding RNAs (lncRNAs). These lncRNAs have been found to be upregulated in cancers and to be essential components of stem cell biology. By studying the role of lncRNAs in the creation of new neurons, we hope to unearth previously unappreciated players in nervous system development and disease.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Predoctoral Individual National Research Service Award (F31)
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Special Emphasis Panel (ZRG1-F08-A (20))
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Owens, David F
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University of California San Francisco
Schools of Medicine
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Ramos, Alexander D; Andersen, Rebecca E; Liu, Siyuan John et al. (2015) The long noncoding RNA Pnky regulates neuronal differentiation of embryonic and postnatal neural stem cells. Cell Stem Cell 16:439-47