Gene Regulatory Networks and Neurodegenerative Diseases Anne Chiaramello, Ph.D Associate Professor...
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Transcript of Gene Regulatory Networks and Neurodegenerative Diseases Anne Chiaramello, Ph.D Associate Professor...
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
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
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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