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

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-DependentNeuronal Differentiation

Undifferentiated Neural Progenitor Cells Differentiated

Neurons

Flowchart to Analyze NeuroD6-Mediated Neuronal Differentiation/Survival

GeneChip Affymetrix Microarray

Functional Analysis ofNeuroD6 Target Genes

Promoter Analysisof NeuroD6 Target Genes

Identification ofRegulatory Elementsand Associated SNPs

Silencing of Target

Genes(siRNA)

Flow Cytometry/Cell Death Assays

Constructing NeuroD6-mediated

Transcriptional RegulatoryNetwork

Ab Initio and Experimental Approaches

Correlation between Altered Gene Expressionand Susceptibility for

Neurodegenerative Diseases

Identification of NeuroD6-Regulated Target Genes During Neuronal Survival by GeneChip Affymetrix

Microarray

Y-axis: GC-RMA: LogRatio (12-13-06), Default InterpretationColored by: Nex1-serumGene List: 1-Way ANOVA (12-12-06) (6059), 17 genes selected

control Nex1+serum Nex1-serum

conditions0.01

0.1

1

10

100

control Nex1+serum Nex1-serum

conditions0.01

0.1

1

10

100

PC12 PC12-ND6

SerumremovalNormal growth conditions

PC12-ND6

1-Microarray analysis does not directly reveal the regulatory networks that underliethe observed transcriptional module mediated by NeuroD6. Combining promoter analysis with microarray results can shed light on NeuroD6-regulated networks2-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

DPEInr+1

TATA

-2000 bp +28-32 bp-35-25 bp

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

Phylogenetic Analysis of the NeuroD6 Promoter

UCSC Genome Browser

P2P1

SNPs

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

E5Hes1Ets2Ets1Hes1MEF2E6E7 C/EBPCdx1 Cdx1

AP1

Sp1E4 E3 E2 E1Hes1Ets2

MEF2

Ets1

HoxD NFB

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