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UNIVERSITY OF CALIFORNIA, SAN DIEGO Regulation of Target Gene Expression in Sensory Neurons by Brn3a A Dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Biomedical Sciences by Jason James Lanier Committee in charge: Professor Eric Turner, Chair Professor Jerold Chun Professor Joseph Gleeson Professor William McGinnis Professor Bing Ren 2007 UMI Number: 3246882 UMI Microform 3246882 Copyright 2007 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.

Box 1346 Ann Arbor, MI 48106-1346 Copyright Jason James Lanier, 2007 All rights reserved The Dissertation of Jason James Lanier is approved, and it is acceptable in quality and form for publication on microfilm. _________________________________________________________ _________________________________________________________ _________________________________________________________ _________________________________________________________ _________________________________________________________ Chair UNIVERSITY OF CALIFORNIA, SAN DIEGO 2007 iii TABLE OF CONTENTS Signature page. iii Table of contents. iv List of Figures.

vi List of Tables. 1 Induction and development of the nervous system. 1 Tissue-specific transcription factors in neural development. 3 Mechanisms of transcriptional regulation.

5 Transcription factor target gene specificity. 7 Epigenetic influence on transcription factor binding. 10 Brn3a is a critical regulator of sensory neural development. Coordinated regulation of gene expression by Brn3a in the developing trigeminal ganglion.

24 Materials and methods. Brn3a target gene recognition in embryonic sensory neurons. 68 Materials and methods. POU-domain factor Brn3a regulates both distinct and common programs of gene expression in the spinal and trigeminal sensory ganglia.

133 Materials and methods. 166 Programs of gene regulation in the nervous system. 166 Direct vs indirect regulation. 167 Context-dependent binding and activity of transcription factors.

169 Cell-specific functions of transcription factors. 175 v LIST OF FIGURES Chapter 2 Figure 2.1 Expression array analysis of E13.2 Brn3a regulates sensory neurotransmitter systems.3 Coordinated regulation of transcription factor expression in sensory ganglia by Brn3a.4 Cellular expression of Brn3a target genes in the CNS.1 Cellular expression of Brn3a in the embryonic trigeminal ganglion. Brn3a is a direct regulator of the NeuroD4 gene in the embryonic trigeminal ganglion.3 ChIP analysis of Brn3a binding to the NeuroD1 and Msc loci in E13.4 Brn3a binding to the Pou4f1 locus in vitro and in embryonic trigeminal ganglia.5 Markers of chromatin modification at the Pou4f1 locus.6 Acetylated histone H3 ChIP assays of the NeuroD4 and NeuroD1 loci.7 Analysis of Brn3a binding sites identified in the promoters of genes not expressed in the developing trigeminal ganglion. 112 vi Chapter 4 Figure 4.1 Analysis of global gene expression in embryonic neural tissue.2 Selective expression of proprioceptor markers in the DRG and trigeminal system.3 Target genes with increased expression in the DRG of Brn3a null mice.4 Target genes with decreased expression in the DRG of Brn3a null mice.5 Brn3a regulation of Hox gene expression in the DRG.6 Trigeminal-specific targets of Brn3a regulation.7 Acetyl-histone H3 profiling of Brn3a target gene loci.

154 vii LIST OF TABLES Chapter 2 Table 2.1 Increased transcripts in the trigeminal ganglion of Brn3a mutant mouse embryos.2 Decreased transcripts in the trigeminal ganglion of Brn3a mutant mouse embryos.3 Significantly changed ESTs in Brn3a mutant mice.4 Relative expression of previously reported Brn3a target genes in the trigeminal ganglia of Brn3a mutant embryos.1 Transcripts differentially expressed in the DRG and TG.2 Increased transcripts in the DRG of Brn3a knockout mice.3 Transcripts decreased in the DRG of Brn3a null mice. 159 viii ACKNOWLEDGEMENT First of all, I would like to thank my advisor, Dr. Eric Turner for being an exceptional mentor and devoting enormous amounts of time and effort to my training. Thanks for all of your guidance and patience and for always having my best interest at heart.

I would also like to thank the members of the Turner lab that I have had the privilege to work with: Natasha, Raisa, Lely, Eric Cox, Iain, and Amanda. You have all helped to create a great working environment in the lab. I have enjoyed getting to know all of you and appreciate your enormous contributions to my thesis project. Finally, I would like to thank the members of my thesis committee for helpful comments and advice along the way.

The text in Chapter Two is a reprint of the material as it appears in Development, 2003, Eng SR, Lanier J, Fedtsova N, Turner EE. I was a secondary author and participated in the research that forms the basis of this chapter. The text of Chapter Three is a reprint of the material as it appears in Developmental Biology, 2007, Lanier J, Quina LA, Eng SR, Cox E, Turner EE. I was the primary researcher, and the co-authors contributed to the research that forms the basis of this chapter.

The text of Chapter Four is a reprint of the material as it appears in Neural Development, 2007, Eng SR, Dykes I, Lanier J, Fedtsova N, and Turner EE. I was a secondary author and participated in the research that forms the basis of this chapter. ix VITA EDUCATION Ph., Biomedical Sciences University of California San Diego School of Medicine 2007 B., Biological Chemistry, cum laude Tulane University 2001 RESEARCH EXPERIENCE Eric Turner Lab, Department of Psychiatry University of California San Diego, La Jolla, CA 2001-Present Larry Byers Lab, Department of Chemistry Tulane University, New Orleans, LA 2000-2001 Tom Wilke Lab, Department of Pharmacology University of Texas Southwestern Medical Center, Dallas, TX 2000 TEACHING EXPERIENCE Salk Institute Mobile Science Lab 2002-2003 General Chemistry Laboratory Course, Tulane University 2000-2001 HONORS UT Southwestern Medical Center Summer Undergraduate Research Fellowship 2000 Reuben Zarilli Memorial Scholarship for Academic Achievement 2000 Conference USA Commissioner’s Academic Medal- Tulane Football 1998 x PUBLICATIONS Eng SR, Dykes I, Lanier J, Fedtsova N, Turner EE (2007). “POU-domain factor Brn3a regulates both distinct and common programs of gene expression in the spinal and trigeminal sensory ganglia.

Lanier J, Quina LA, Eng SR, Cox E, Turner EE (2007). “Brn3a target gene recognition in embryonic sensory neurons.” Developmental Biology, 302(2) pp. Cox E, Lanier J, Quina L, Eng SR, Turner EE (2006) “Regulation of FGF10 by POU transcription factor Brn3a in the developing trigeminal ganglion.” Journal of Neurobiology, 66(10):1075-83. Quina LA, Pak W, Lanier J, Banwait P, Gratwick K, Liu Y, Velasquez T, O’Leary DD, Goulding M, Turner EE (2005).

“Brn3a-expressing retinal ganglion cells project specifically to thalamocortical and collicular visual pathways.” Journal of Neuroscience, 25(50):11595-604. Eng SR, Lanier J, Fedtsova N, Turner EE (2004). “Coordinated regulation of gene expression by Brn3a in developing sensory ganglia. ABSTRACTS Lanier J, Quina L, Turner EE (2006).

“Tissue-specific regulation of gene expression by Brn3a.” Keystone Symposium- Regulation of Eukaryotic Transcription: From Chromatin to mRNA, Taos, NM. Lanier J, Quina L, Cox E, Turner EE (2005). “Locus-wide ChIP analysis of the transcriptional targets of Brn3a in the sensory ganglia.” Society for Neuroscience Annual Meeting, Washington, DC. Quina L, Lanier J, Eng S, Banwait P, Liu Y, Goulding M, Turner EE (2004).

“Role of Brn3a in CNS axon guidance.” Society for Neuroscience Annual Meeting, San Diego, CA. Lanier J, Eng SR, Fedtsova N, Turner EE (2003). “Transcriptional targets of Brn3a in the sensory ganglia.” Society for Neuroscience Annual Meeting, New Orleans, LA. xi ABSTRACT OF THE DISSERTATION Regulation of Target Gene Expression in Sensory Neurons by Brn3a by Jason James Lanier Doctor of Philosophy in Biomedical Sciences University of California, San Diego, 2007 Professor Eric Turner, Chair During the development of the vertebrate nervous system, cellular proliferation and differentiation result in the formation of a large number of specialized physical structures composed of many different types of cells.

The phenotypic properties of a cell are largely controlled by the complement of proteins that the cell expresses. Thus, the formation of properly functioning neuronal circuitry requires precisely coordinated regulation of gene expression. Tissue-specific transcription factors play a critical role in the process of development by exerting spatiotemporal control over the expression of their specific target genes. Hundreds of transcription factors have been identified xii which exhibit tissue-specific expression in the nervous system.

Many of these factors are critical for proper neural development, with their disruption leading to severe phenotypic abnormalities. Brn3a (Pou4f1) is a POU domain transcription factor with a highly specific and complex pattern of expression in the developing nervous system. Disruption of Brn3a function results in severe developmental abnormalities and neonatal lethality. We have identified programs of gene expression controlled by Brn3a in both the trigeminal ganglia (TG) and dorsal root ganglia (DRG) using microarray analysis of dissected tissue from wild-type and Brn3a null embryos in midgestation.

These experiments indicate that Brn3a regulates similar, but distinct complements of transcripts in sensory ganglia at different axial levels. We then show, using in vivo chromatin immunoprecipitation (ChIP) assays, that Brn3a directly represses the expression of the neurogenic transcription factors NeuroD1 and NeuroD4 in the developing TG. Finally, we provide evidence that epigenetic modifications of chromatin play an integral role in determining the regulatory targets of Brn3a and contribute to target gene differences between the TG and DRG. The work presented in this dissertation has provided insight into the role of Brn3a in the developing sensory nervous system.

It also demonstrates a clear role for chromatin modification in transcription factor target gene selection which may be relevant for many tissue- specific transcription factors. xiii I Introduction Induction and development of the nervous system The induction and development of the vertebrate nervous system comprise an extraordinarily complex series of events resulting in the generation of a multitude of distinct cell types and structures (Kandel, Schwartz et al. During early mammalian embryonic development, prior to the induction of the nervous system, the embryo consists of three main cell layers. The innermost cell layer, the endoderm, consists of cells that eventually give rise to the gut and internal organs.

The vascular system, musculature, and connective tissues are derived from the middle cell layer, the mesoderm. The major tissues of the central and peripheral nervous systems are generated from the ectoderm, located on the surface of the early embryo. At the gastrula stage of development, a sheet of cells located at the dorsal midline of the embryo, begins to acquire neural properties and forms a structure called the neural plate, which is the source of both neural and glial cells. In a process called neurulation, the neural plate folds into a structure called the neural tube, which eventually gives rise to the central nervous system.

The adult spinal cord and brain are derived from the posterior and anterior regions of the neural tube, respectively (Schoenwolf, Bortier et al. 1989; Eagleson and Harris 1990). 1 2 Many of the cells comprising the peripheral sensory nervous system are derived from a group of migratory cells called the neural crest (Selleck and Bronner- Fraser 1996). Prior to neurulation, precursors of this specialized group of cells can be identified at the border of the neural plate and the non-neural ectoderm (Huang and Saint-Jeannet 2004).

Around the time of neural tube closure, neural crest cells migrate ventrally throughout the embryo, differentiating into a wide range of both neuronal and nonneuronal cells, including a majority of sensory neural progenitors. The remainder of the cells in the peripheral sensory nervous system originate in ectodermal cellular structures called neurogenic placodes (Barlow 2002). The patterning and differentiation of cells in the nervous system is ultimately controlled by signaling molecules called inducing factors which are secreted from cells in a particular location within an embryo and influence the physiology of surrounding cells. Secretion of an inducing factor from a localized region within an embryo creates a signaling gradient which can determine the arrangement and fate of responding cells according to the concentration of the factor perceived by each cell (Gurdon and Bourillot 2001).

Cells occupying different positions within a developing embryo are exposed to different inducing factors. Thus the position that a cell occupies early in development has a direct influence over its ultimate fate.