Daniel A. Lim, M.D., Ph.D. - US grants

DESCRIPTION (Provided by the applicant) Abstract: This proposal addresses two fundamental questions at the crossroads of epigenetics, stem cell biology, and regenerative medicine that relate to the chromatin-based cellular memory system. 1. Do chromatin modifications at specific genetic loci predict the progressive "restriction" of differentiation potential that occurs in neural stem cells during brain development and into adulthood? 2. Can cellular memory systems be partially "erased" or "reset" at the chromatin level in precursor cell populations to broaden their developmental potential? These studies should greatly advance our understanding of how precursor cells "remember" both their temporal and positional identities as well as determine whether this cellular memory system can be manipulated for novel therapeutic strategies. Three areas of impact are: Developmental Neurobiology, where results shed light on epigenetic mechanisms of neuronal and glial differentiation;Regenerative Medicine, where insight gained may suggest novel methods of cell fate specification;and Cancer Biology, where results may reveal how certain chromatin derangements can promote brain tumors. First, we propose investigating the changes in chromatin modifications that occur along a neural stem cell continuum from the embryo and into adulthood. Our proposed methods utilizing cells acutely isolated from the brain represent a significant advancement upon current cell culture based studies. To accomplish this, we must innovate new, integrative approaches for chromatin study. We will also employ novel and as of yet unproven approaches to "reset" chromatin memory of cell identity with the purpose of altering cell fate. Given that these ideas concerning the chromatin basis of cellular memory and strategic epigenetic manipulation are new and relatively untested, the level of risk in our proposal is substantially higher than in traditional investigator grants. We wish to embark on this tangent from our current studies to broaden the impact of our research and explore fundamental principles of cellular memory in stem cell biology. Public Health Relevance: Neural stem cells hold promise for the treatment of neurological disorders, and understanding the epigenetic mechanisms by which these precursors differentiate into neurons and glia may be key to unlocking their therapeutic potential. By performing research at the crossroads of epigenetics, stem cell biology, and regenerative medicine, we may develop novel methods of "engineering" cells for cell replacement strategies. Furthermore, our studies may lead to discoveries of how derangements in chromatin remodeling can lead to the development of brain tumors.

? DESCRIPTION (provided by applicant): Long noncoding RNAs (lncRNAs) - transcripts longer than 200 nucleotides with little evidence of protein coding potential - have been implicated in a wide range of human neurological disorders including cancer, developmental delay, schizophrenia and Alzheimer's disease. While the mammalian genome has been discovered to transcribe many thousands of lncRNAs, very few lncRNAs have been characterized in terms of in vivo function and molecular mechanism. In a genome-wide analysis of lncRNAs in adult ventricular- subventricular zone (V-SVZ) neurogenesis, we identified a novel lncRNA transcript named Pinky (Pnky). We have recently demonstrated that Pnky regulates the production of neurons from NSCs of the embryonic and postnatal brain. Pnky is a neural-specific, nuclear lncRNA transcript. In the V-SVZ neurogenic lineage, Pnky is expressed in NSCs and becomes downregulated during neuronal differentiation. In postnatal V-SVZ NSCs, Pnky knockdown potentiates neuronal lineage commitment and expands the transit-amplifying cell population, increasing neuron production several-fold. Pnky is evolutionarily conserved and expressed in NSCs of the developing human brain. In the embryonic mouse cortex, Pnky knockdown increases neuronal differentiation and depletes the NSC population. Mass spectrometry, Western blot, and RNA immunoprecipitation analysis indicates that Pnky physically interacts with PTBP1, a known regulator of neurogenesis, brain tumors, direct cell reprogramming, and RNA splicing. In NSCs, Pnky and PTBP1 regulate the expression and alternative splicing of a core set of transcripts that relates to the cellular phenotype. We have since generated a Pnky conditional knockout (Pnky-cKO) mouse, and this genetic model of Pnky-deficiency phenocopied Pnky knockdown both in vitro and in vivo. The overall goal of the proposed work is to understand the in vivo function and mechanism of Pnky. Aim 1 is to determine the role of Pnky in adult V-SVZ neurogenesis by studying Pnky-deficiency and Pnky transgenic expression in vivo. Preliminary Data, our expertise in V-SVZ biology, and the use of multiple, complementary approaches for manipulating Pnky expression support the feasibility of Aim 1. Aim 2 is to determine the mechanism(s) by which Pnky regulates neurogenesis. Whether Pnky and PTBP1 functionally interact will be investigated with the analysis of biological phenotypes, transcriptome changes, and RNA-protein interactions. The discovery of additional factors that interact with Pnky will provide the basis for investigating other potential lncRNA mechanisms. In addition to Preliminary Data, collaborations with Dr. Aaron Diaz (bioinformatics), Dr. Nevan Krogan (mass spectrometry), Dr. Seth Blackshaw (protein microarrays), and Dr. Hiten Madhani (RNA splicing, RNA-protein interactions) support the feasibility of Aim 2. Such knowledge of lncRNA developmental and mechanistic function will provide critical insight into how lncRNAs can underlie neurological disease and may inform the development of lncRNAs as therapeutic targets.

ABSTRACT The human genome produces many thousands of long noncoding RNAs (lncRNAs) ? transcripts >200 nucleotides long with little evidence of protein coding potential. It is now clear that lncRNAs can have critical biological functions and roles in human disease including cancer. Because lncRNAs are particularly cell and disease specific, they are attractive as therapeutic targets. However, our understanding of lncRNAs in primary brain tumors is still very limited. Glioblastoma multiforme (GBM) is the most common primary malignant brain tumor. Despite surgery, chemotherapy and radiation treatment, the median survival after diagnosis of GBM is only 14-16 months. Our long-term goal is to develop highly specific and effective new therapies for the treatment of primary brain tumors including GBM. In pursuit of this goal, our immediate objective is to identify and pursue specific lncRNAs as therapeutic targets in human gliomas. CRISPR interference (CRISPRi) is a readily scalable and highly specific technology for transcriptional regulation, and we have recently implemented this as a method for lncRNA knockdown in human GBM cells. In Preliminary Studies, we have developed CRISPRi for large-scale, genome-wide screening of lncRNA function in human brain tumor cells. In our pilot screen of ~1300 GBM-expressed lncRNAs, we identified 27 that regulate the propagation of GBM cells in culture. CRISPRi screen ?hits? could be individually validated with both CRISPRi and antisense oligonucleotide (ASO) mediated knockdown. Knockdown of one particular lncRNA ? LINC00909 ? strongly reduced glioma cell propagation in culture and in a xenograft mouse model. LINC00909 overexpression is very specific to GBM tumors, and higher LINC00909 expression predicts shorter survival of patients with mesenchymal GBM. Interestingly, in the developing human brain, LINC00909 was enriched in normal neural stem cells, and expression analysis suggests LINC00909 expression in GBM cancer stem cells (CSCs). Given these Preliminary Studies, our central hypothesis is that specific lncRNAs such as LINC00909 are uniquely required for GBM tumor growth but not the viability of normal adult human brain cells. In this proposal, our first Aim is to determine the role of LINC00909 in models of human GBM both in vitro and in vivo. Whether LINC00909 has essential function in normal glia and neurons will also be tested. For our second Aim, we will use CRISPRi to more comprehensively identify lncRNAs that regulate GBM tumor growth. In addition to further testing our central hypothesis, this work will provide an important data resource (lncRNA hit identification) and novel tools (large-scale CRISPRi screening libraries and methods) ? both of which we will make available for distribution. In addition to our expertise with lncRNAs and Preliminary Studies, our ongoing local collaborations with Dr. Jonathan Weissman (CRISPRi), Dr. Aaron Diaz (bioinformatics) and Dr. Nalin Gupta (primary human brain tumor models) support the feasibility of this work. By accomplishing these studies, we will lay novel, important groundwork for the development of lncRNAs as targets for glioma therapy.

JMJD3 (KDM6B) is a chromatin regulator with roles central to normal development as well as a wide range of human diseases including cancer and human neurological disorders. For instance, mutations in JMJD3 are autosomal recessive for familial intellectual disability, and de novo JMJD3 mutations are associated with autism spectrum disorder (ASD). An important next step to understanding genetic causes of complex diseases is to study disease-associated genes in mouse models. While it is known that JMJD3 is important to certain aspects of neural cell development, whether JMJD3 deficiency can actually cause cognitive dysfunction has not been known. Preliminary Studies indicate that JMJD3 is critical for the development of the mouse hippocampal dentate gyrus (DG). In the DG, granule neurons are generated throughout life from a population of neural stem cells (NSCs). Defective DG neurogenesis impairs many hippocampal-dependent behaviors and has been associated with cognitive deficits including that of intellectual disability and ASD. Without Jmjd3, NSCs failed to become established in the adult DG, and granule neuron production was severely decreased and abnormal. In these mice, hippocampal-dependent learning was defective. Heterozygous deletion of Jmjd3 also resulted in abnormal postnatal DG development, indicating that this process is sensitive to gene dosage. Aim 1 is to determine the role of Jmjd3 in DG neurogenesis. In vivo experiments will test the hypothesis that Jmjd3 regulates the postnatal expansion and establishment of the DG NSC population, and that even reduced Jmjd3 gene dosage causes cognitive dysfunction. Single cell RNA sequencing analysis will provide molecular insights into the observed phenotype and help guide mechanistic studies of Aim 2. Aim 2 is to determine the mechanisms by which JMJD3 regulates gene expression. JMJD3 has demethylase activity for histone 3 lysine 27 trimethylation (H3K27me3), which is a chromatin modification associated with transcriptional repression. To investigate demethylase-dependent and potential demethylase-independent activities of JMJD3, we have developed innovative CRISPR-based technologies to recruit JMJD3 proteins to the genome. By developing a novel, easy-to-use method for mapping lamina-associated domains (LADs) ? a repressive nuclear compartment ? we have also found that JMJD3 in NSCs is enriched at the genomic LAD ?borders,? which are genomic regions enriched for transcriptional regulatory elements. Thus, we propose investigating the role of JMJD3 in regulating this aspect of higher-order chromatin structure. The proposed neurodevelopmental and behavioral analyses combined with mechanistic studies is expected to provide a scientific framework in which to begin understanding how human JMJD3 mutations can cause disease. The studies of JMJD3 mechanism is also expected to be important to the broader field of chromatin-based epigenetics as well as nuclear compartment-associated genome organization ? an emerging area of research.