Analysis of Promoter Shifting
Using CAGE data
An insight into transcription regulation
11/11/2014
Jack Binysh MRC Internship
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Outline
• Background
 Introduction to Promoters and Transcription Start Sites (TSS’s)
 Classification of Promoters
 Motivation for Project
• Project
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11/11/2014
CAGE data
Previous work
CAGEr
Results
Future Work
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Promoters and TSS’s
How is regulation achieved?
• Promoter region
 Contains regulatory
elements (binding motifs,
CG enrichment…)
 Controls gene expression
• Context specific
• Dynamic
• Associated epigentics
 Histone placement
 Histone ‘marks’
11/11/2014
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Classification
• Correlation between
several features
 Broad vs Sharp
 CG islands vs TATA
 Ordered vs Disorded
Histones
 General vs Specific
function
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CAGE Data
• Cap Analysis of Gene Expression
• mRNA captured, first ~20 bp
sequenced from 5’ end  Tags
 Full length estimated
 Tags mapped to Genome
• TSS determination at bp resolution.
• Genome wide mapping of mRNA
transcription
• FANTOM5 – CAGE datasets
for many cell types
A ‘TSS Profile’
11/11/2014
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Motivation for Project
• Already known that:
 One Gene may have multiple types of promoter → regulated in
several ways
 Variants of Transcription factors may exist in different cells
Do the rules governing transcription change
between cell types?
Both temporally (embryonic development) and spatially (in adult tissues) ?
•Focus on housekeeper genes – always expressed
•Look for changes in TSS profile between cell types…
11/11/2014
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CAGEr
Input
Output
Available
resources
Methods
TSS
Tag
clusters
(TC)
Normalized
expression
Custom input
CAGEset
CAGE bam
files
CTSS files
11/11/2014
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Clustering in CAGEr
• Two levels of clustering
 TSS profiles  Tag clusters
 Tag cluster  Consensus
•Tag clusters sample specific
•Consensus clusters the
same for all samples
TCs
CTSSs
S1
S2
S3
TCs
consensus cluster
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Extending CAGEr
•Large datasets
• 1 sample ~ 46 million tag sites
•FANTOM5 has hundreds of samples
•Pairwise comparisons of datasets O(n2)
• if 1 comparison takes ~ 1 hour, 60
samples takes ~ 10 weeks!
•Need to speed things up, avoid doing every
comparison, etc.
11/11/2014
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Dendrogram
•67 cell types compared
•Most show very little
shifting –’bulk’
•~ 8 ‘outliers’
Cardiac Myocytes
Sertoli Cells
Hepatocytes
Hair follicle papilla
CD 19
Renal Glomerular
Neurons
Aortic Endothelial
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Heatmap
•Each outlier is separated from
every other cell type
•The difference between two
outliers is greater than the
difference between one outlier
and the ‘bulk’
•Suggests a different set of
shifting promoters in every
outlier
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Scatter plots
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Dinucleotide Density plots
• Each cluster has two
dominant TSS’s – modal
and sample specific
Cardiac Myocytes
Centered on modal TSS
• Look for dinucleotide
enrichment in sequences
• Initiator sequence at
modal CTSS visible
Cardiac Myocytes
Centered on outlier TSS
• No obvious motif at the
TSS of the outlier
11/11/2014
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Motif discovery
• Motif discovery finds no specific motifs 500 bp either side of the outlier
TSS in any of the samples.
• All of the samples show general GC enrichment
• ~80% clusters overlap 1 annotated CpG island, ~20% overlap none
Cardiac Myocytes
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Gene Ontology
•
Gene Ontology analysis
 Each cluster associated with nearest annotated TSS & entrezgene ID
 Keywords tagged to each entrezgene ID
 Statistics on over/under representation of Keywords
Cardiac Myocytes
11/11/2014
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Gene Ontology
•Significantly over-represented Biological functions tend to be housekeeping –
not cell specific
•Perhaps the shifting promoters are not involved with cell specific gene function
at all?
Cardiac Myocytes
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Future Work
• Repeat analysis using different consensus clusters
 Problems with thresholds within analysis
 More promising recent dinucleotide maps
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Future Work
• Analysis of non-shifting promoters
 Looking at more general changes in shape
 Eg . Dot product, linear scaling
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Extra slides…
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Previous Results
•Zebrafish embryonic development
•Initial RNA transcriptome inherited from
mother, zygotic gene activation at Mid Blastula
Transition
•Corresponds to change in TSS profile
Sharp Broad
Position of TSS’s shift
Shifting Promoters
•“Differential promoter interpretation by the
maternal and zygotic transcription
machinery”
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Shifting Promoters
Search for genetic structure
correlated with this shifting
•TATA like enrichment always found
~30 bp upstream in Maternal
•In Zygote, boundary 50 bp
downstream of TSS
•Majority of TATA- like motifs not
canonical TATA boxes (W box)
Two Independent Mechanisms
for Transcription Initiation
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Nucleosome Location
•H3K4me3 Nucleosome locations
estimated at 4 developmental stages
•Alignment with Zygotic, but not
Maternal, TSS, 50bp downstream
 Same location as boundary
• Suggests Zygotic mechanism for
positioning nucleosomes after MBT
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Internucleosomal Phasing Patterns
•10 bp AA/TT dinucleotide enrichment
periodicity downstream of zygotic
TSS, but not maternal
•Weaker GC/AT enrichment pattern
matching nucleosome free and
wrapped DNA
•Zygotic,not maternal, TSS associated
with nucleosome positioning
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