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Georg Gerber, PhD Gifford Laboratory, MIT CSAIL April 9, 2009

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Initial Steps Toward Computational Discovery of Genetic Regulatory Networks in Pancreatic Islet Development

Georg Gerber, PhD

Gifford Laboratory, MIT CSAIL

April 9, 2009

- Goals
- Expression data overview
- TF-TF interaction networks
- pair-wise mutual information
- Bayesian networks

- Gene expression programs
- ChIP-seq data
- Directions for future work

- Knowledge of distinct signaling/transcriptional steps involved in pancreatic specification
- Optimize ES differentiation by determining signaling event(s) directly inducing each sequential TF

- What is the network structure? Linear or cross-regulatory, parallel or all interrelated
- Direct reprogramming using TFs would benefit from knowing hierarchy of each network
- Are TFs that play role in specification of pancreas necessary for later function of pancreas or are they merely required to properly induce other necessary TFs?

- Can knowledge of the pancreatic specification network teach us about lineage diversification within the pancreas (endocrine, exocrine, duct)?

- Determine set of transcription factors active at different developmental stages
- Discover network “wiring”
- Determine how network changes/evolves throughout development
- Compare in vivo and ESC networks

- Goals
- Expression data overview
- TF-TF interaction networks
- pair-wise mutual information
- Bayesian networks

- Gene expression programs
- ChIP-seq data
- Directions for future work

E8.25

Embryonic ectoderm/notochord

Embryonic mesoderm

Definitive endoderm

(E7.75 and E8.75 as well)

E11.5

Stomach

endoderm

Intestinal

endoderm

Pancreatic

Endoderm

(E10.5 as well)

Lung

endoderm

Liver

endoderm

Esophageal

endoderm

Tcf2

Foxa2

DMSO

DMSO/

2 uM RA

6h/24h

50 ng/mLActA

6 days

ES

Sox17

GFP+

FACS sort Sox17GFP+Dpp4- definitive endoderm

and perform microarray

2 uM RA

- Implant bead coated with DMSO/RA into foregut of E8.25 (4-6 somite) embryo
- Explant embryo anterior to 1stsomite
- Culture for 6/24 hours
- Dissociate, sort for EpCAM+ endoderm
- Amplify RNA and profile on Illumina Mouse Ref8 v2 chips

- 120 Illumina arrays (18118 genes/array)
- 72 distinct experiments (41 in mESC’s)
- Standardized mESC/in vivo experiments separately
- 2758 genes w/ ≥ 2-fold change in ≥ 5 experiments
- 154 TFs w/ ≥ 2-fold change in ≥ 5 experiments (out of 946 “definite” or “candidate” TFs from TFCat, Fulton et al, Genome Biology 2009)

- Need 100’s of varied experiments for finding relevant/significant networks
- Association ≠ causation
- High false positive rates (high dimensional, noisy, dependent data)
- High false negative rates (low TF transcript abundance, post-transcriptional regulation, etc.)

- Goals
- Expression data overview
- TF-TF interaction networks
- pair-wise mutual information
- Bayesian networks

- Gene expression programs
- ChIP-seq data
- Directions for future work

- Context Likelihood of Relatedness method: Faith et al., PLoS Biology 2007
- Computes MI between all genes
- Innovation: considers MI distribution for both target and source to compute p-values/estimate FDR

TF-TF network (MI)

E8.25 4-6s definitive endoderm

TF-TF network (MI)

E8.75 13-15s definitive endoderm

TF-TF network (MI)

E9.5 definitive endoderm

TF-TF network (MI)

E10.5 pancreatic endoderm

TF-TF network (MI)

E11.5 pancreatic endoderm

TF-TF network (MI)

E11.5 intestinal endoderm

TF-TF network (MI)

6h 83 uM RA bead

mES 2 uM RA 6h

TF-TF network (MI)

24h 83 uM RA bead

mES 2 uM RA 24h

- Goals
- Expression data overview
- TF-TF interaction networks
- pair-wise mutual information
- Bayesian networks

- Gene expression programs
- ChIP-seq data
- Directions for future work

- Directed networks, allow for multiple parents
- Encode conditional independence
- Penalize complexity automatically
- Software: Banjo (Alexander Hartemink, Duke University)

E8.25 4-6s definitive endoderm

TF-TF network (Bayes Net)

E8.75 13-15s definitive endoderm

TF-TF network (Bayes Net)

E9.5 definitive endoderm

TF-TF network (Bayes Net)

E10.5 pancreatic endoderm

TF-TF network (Bayes Net)

E11.5 pancreatic endoderm

TF-TF network (Bayes Net)

mES 2 uM RA 6h

6h 83 uM RA bead

TF-TF network (Bayes Net)

mES 2 uM RA 24h

24h 83 uM RA bead

TF-TF network (Bayes Net)

- Goals
- Expression data overview
- TF-TF interaction networks
- pair-wise mutual information
- Bayesian networks

- Gene expression programs
- ChIP-seq data
- Directions for future work

- Infer more robust relationships because considering many genes
- Allow for enrichment analysis
- Functional categories
- Signaling pathways
- TF DNA binding sequence motifs

- Gerber et al, PLoS Comp Bio 2007
- Discovers sets of genes co-expressed across subsets of conditions
- Innovations:
- Simultaneously models probabilistic structure of experiments (tissues) and genes
- Uses Hierarchical Dirichlet Processes, a fully Bayesian method for automatically determining the number of expression programs and tissue groups
- Outperforms state-of-the-art biclustering methods

Hierarchical clustering

Singular Value Decomposition (SVD)

Non-negative Matrix Factorization (NMF)

GeneProgram w/o tissue groups

Full GeneProgram model

tissue groups

GeneProgram produced a map of 12 tissue groups and 62 expression programs

tissue

GeneProgram produced a map of 12 tissue groups and 62 expression programs

GeneProgram produced a map of 12 tissue groups and 62 expression programs

expression programs (sorted by generality score)

GeneProgram produced a map of 12 tissue groups and 62 expression programs

expression program use by tissue

- GO categories
- FDR controlled to 5%

- TRANSFAC motifs
- Software: SAMBA
- Scans +3000 to -200 bp for each motif
- Uses PWM to score region, background to calculate p-value (Bonferroni corrected)

Expression programs (GO and motif enrichment)

E8.25 4-6s definitive endoderm

Expression programs (GO and motif enrichment)

E8.75 13-15s definitive endoderm

Expression programs (GO and motif enrichment)

E9.5 definitive endoderm

Expression programs (GO and motif enrichment)

E10.5 pancreatic endoderm

Expression programs showing TFs in programs and motif enrichment

E8.25 4-6s definitive endoderm

Expression programs showing TFs in programs and motif enrichment

E8.75 13-15s definitive endoderm

Expression programs showing TFs in programs and motif enrichment

E9.5 definitive endoderm

Expression programs showing TFs in programs and motif enrichment

E10.5 pancreatic endoderm

Expression programs showing TFs in programs and motif enrichment

E11.5 pancreatic endoderm

- Goals
- Expression data overview
- TF-TF interaction networks
- pair-wise mutual information
- Bayesian networks

- Gene expression programs
- ChIP-seq data
- Directions for future work

- Generated in the Wichterle lab at Columbia (unpublished data, Motor Neuron Development Project)
- mESC’s grown to embryoid body stage, profiled after 8h of RA exposure

Binding events determined with modified MACS method (Zhang et al, Genome Biology 2008); called if significant peak found w/in 50 kb of gene start site

- Add publically available ES expression data
- Apply more sophisticated TF binding motif methods (phylogeny, spatial arrangements, co-regulation)
- Extend GeneProgram framework for add’l data types (TF expression, binding motifs, ChIP-seq, knockdown/overexpression, ?protein-protein interactions, etc.) → causal/predictive models
- Infer dynamic rewiring networks over inferred developmental tree
- Develop novel probabilistic methods for ChIP-seq data

- Rich Sherwood (Melton lab) - all the expression data!
- Arvind Jammalamadaka (Gifford lab) -initial data analysis/normalization methods
- Shaun Mahony (Gifford lab) - RA ChIP-seq data analysis
- Esteban Mazzoni (Wichterle lab) - RA ChIP-seq data

TF-TF network (MI)

E11.5 stomach endoderm

TF-TF network (MI)

E12.5 esophagus endoderm

TF-TF network (MI)

E11.5 liver endoderm

TF-TF network (MI)

E11.5 lung endoderm

E8.25 anterior endoderm

E8.25 4-6s ectoderm

E8.25 4-6s mesoderm

6h 83 mM RA bead

d1 83 mM RA bead

mES 2mM RA 6h

mES 2mM RA 24h

mES differentiated 7d

N = Novartis Tissue Atlas v2 (141 mouse and human tissues)

S = Shyamsundar et al. (115 human tissues)