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-Dependent
Neuronal Differentiation
Undifferentiated Neural Progenitor Cells
Differentiated Neurons
Flowchart to Analyze NeuroD6-Mediated Neuronal Differentiation/Survival
GeneChip Affymetrix Microarray
Promoter Analysis
of NeuroD6 Target Genes
Functional Analysis of
NeuroD6 Target Genes
Constructing
NeuroD6-mediated
Transcriptional Regulatory
Network
Silencing of Target
Genes(siRNA)
Flow Cytometry/Cell
Death Assays
Ab Initio and
Experimental Approaches
Identification of
Regulatory Elements
and Associated SNPs
Correlation between Altered Gene Expression
and Susceptibility for
Neurodegenerative Diseases
Identification of NeuroD6-Regulated Target Genes During
Neuronal Survival by GeneChip Affymetrix Microarray
100
10
1
0.1
conditions
0.01
control
Y-axis:
Colored by:
Gene List:
Nex1+serum
Nex1-serum
GC-RMA: LogRatio (12-13-06), Default Interpretation
Nex1-serum
1-Way ANOVA (12-12-06) (6059), 17 genes selected
PC12
PC12-ND6
Normal growth conditions
PC12-ND6
Serum
removal
Computational Approaches to Identify the Underlying Transcriptional Network
of Gene Expression from Microarray Analysis.
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.
Core Promoter (-250/+150 bp)
Enhancers
TSS
Proximal Promoter (5’UTR)
Inr
-35-25 bp +1
TATA
-2000 bp
DPE
+28-32 bp
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.
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
P2
P1
SNPs
UCSC Genome Browser
Prediction of Transcription Factor Binding Sites (TFBS)
•TRANSFAC
•JASPAR
•rVista
•Huge numbers of false positive
•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.
-1800
AP1
Cdx1 E7 E6
-750
-1453
MEF2
HoxD NFkB
Cdx1 MEF2 C/EBP Hes1 Ets1 Ets2 Hes1 E5
*
*
* *
6 Sp1 sites
Ets1
E4
E3
* ** *
Sp1 Ets2 Hes1 E2
* *
*
E1
+1