1. Gene Regulatory Networks and Neurodegenerative Diseases Anne Chiaramello, Ph.D Associate Professor George Washington University Medical Center Department of Anatomy and Cell Biology Tel: 202-994-2173 anaaec@gwumc.edu NIH/NINDS R01NS041391 McCormick Pilot Grant January 24, 2007

2. Long-Term Medical Applications of our Research Programs Correlation between Altered Gene Expression and Susceptibility for Specific Neurodegenerative Diseases Genetic Manipulation of Neural Stem/Progenitor Cells to Promote Specific Neuronal Identity and Survival upon Transplantation Overall Strategy To dissect NeuroD6-Mediated gene regulatory networks responsible for Initiation/ Maintenance of differentiation, and neuronal survival. To Identify Dysregulation of Neuronal-Specific Genes Associated with Neurodegenerative Disorders. Transcription-Dependent Neuronal Differentiation Undifferentiated Neural Progenitor Cells Differentiated Neurons

3. Flowchart to Analyze NeuroD6-Mediated Neuronal Differentiation/Survival GeneChip Affymetrix Microarray Functional Analysis of NeuroD6 Target Genes Promoter Analysis of NeuroD6 Target Genes Identification of Regulatory Elements and Associated SNPs Silencing of Target Genes(siRNA) Flow Cytometry/Cell Death Assays Constructing NeuroD6-mediated Transcriptional Regulatory Network Ab Initio and Experimental Approaches Correlation between Altered Gene Expression and Susceptibility for Neurodegenerative Diseases

4. Identification of NeuroD6-Regulated Target Genes During Neuronal Survival by GeneChip Affymetrix Microarray PC12 PC12-ND6 Serum removal Normal growth conditions PC12-ND6

5. 1-Microarray analysis does not directly reveal the regulatory networks that underlie the observed transcriptional module mediated by NeuroD6. Combining promoter analysis with microarray results can shed light on NeuroD6-regulated networks 2-A promoter is defined as a functional region immediately upstream and downstream of a Transcriptional start site (TSS) that is ultimately involved in the regulation of transcription. 3-The putative TSS for 17,702 transcripts corresponding to 13,300 genes have been annotated. Given a correct estimate of ~25,000 human genes, promoters for a majority of genes in the human genome remain to be fully defined. 4-Furthermore, transcriptional regulation of most genes originates from at least two distinct promoters,located in different non-coding exons, with the upstream promoter most of the time unknown. Computational Approaches to Identify the Underlying Transcriptional Network of Gene Expression from Microarray Analysis. Core Promoter (-250/+150 bp) Enhancers Proximal Promoter (5 ’ UTR) TSS DPE Inr +1 TATA -2000 bp +28-32 bp -35-25 bp

6. Ab Initio Methods to Predict Promoter Structure Database of Transcription Start site (db TSS) Cold Spring Harbor Laboratories Mammalian promoter Database Genome Browser: UCSC, ENSEMBL, NCBI Cap Analysis of Gene Expression (Riken, CAGE Data) Promoter Predictions Algorithms GRAIL Exp v3.3 (Gene Recognition and Analysis Internet work) Promo H Algorithm Promoter Inspector Dragon Promoter Finder De novo FIRST EF ( First Exon-Finding) CpG Island (NCBI Map Viewer) Phylogenic Footprinting Analysis: multi-species sequenced conservation (ClustalW and Genome Browsers UCSC, ENSEMBL, NCBI) Experimental Approaches for Promoter Identification 5’RACE Primer Extension Luciferase Reporter-Promoter Assay

7. Phylogenetic Analysis of the NeuroD6 Promoter UCSC Genome Browser P2 P1 SNPs

8. Prediction of Transcription Factor Binding Sites (TFBS) To reduce false positives, focus on: binding sites conserved among conserved species identified by several algorithms. Position Weight Matrix comparison Module Searcher Experimental Verification of TFBS DNaseI Footprinting Analysis/EMSA ChIp Site-direct mutagenesis/reporter-promoter assay. TRANSFAC JASPAR rVista Huge numbers of false positive 6 Sp1 sites -1800 +1 E5Hes1 Ets2 Ets1 Hes1 MEF2 E6 E7 C/EBPCdx1 AP1 Sp1 E4 E3 E2 E1Hes1Ets2 MEF2 Ets1 HoxD NF  B ** ***** ** * * -1453 -750