analysis of promoter shifting using cage data · introduction to promoters and transcription start...

11/11/2014 1Jack Binysh MRC Internship

Analysis of Promoter Shifting

Using CAGE data

An insight into transcription regulation

Outline

• Background

Introduction to Promoters and Transcription Start Sites (TSS’s)

Classification of Promoters

Motivation for Project

• Project

CAGE data

Previous work

CAGEr

Results

Future Work

11/11/2014 Jack Binysh MRC Internship 2

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’


Classification

• Correlation betweenseveral features

Broad vs Sharp

CG islands vs TATA

Ordered vs DisordedHistones

General vs Specificfunction


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’

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…

CAGEr


Input Output

Methods

CAGEset

CAGE bam

files

Availa

ble

resourc

es

Custo

m input

CTSS files

TSS

Tag

clusters

(TC)

Normalized

expression

Clustering in CAGEr

• Two levels of clustering

TSS profiles Tag clusters

Tag cluster Consensus


S1

S2

S3

consensus cluster

TCs

CTSSs

TCs

•Tag clusters sample specific

•Consensus clusters the

same for all samples

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.

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

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

Scatter plots


Dinucleotide Density plots

• Each cluster has two

dominant TSS’s – modal

and sample specific

• Look for dinucleotide

enrichment in sequences

• Initiator sequence at

modal CTSS visible

• No obvious motif at the

TSS of the outlier


Cardiac Myocytes

Centered on outlier TSS

Cardiac Myocytes

Centered on modal TSS

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

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

Gene Ontology


Cardiac Myocytes

•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?

Future Work

• Repeat analysis using different consensus clusters

Problems with thresholds within analysis

More promising recent dinucleotide maps


Future Work

• Analysis of non-shifting promoters

Looking at more general changes in shape

Eg . Dot product, linear scaling


Extra slides…


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”

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

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

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

analysis of promoter shifting using cage data · introduction to promoters and transcription start...

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