Searching for Transcription

Start Sites in Drosophila

Wilson Leung 08/2015

 Outline
Transcription start sites (TSS) annotation goals
Promoter architecture in D. melanogaster
Find the initial transcribed exon
Annotate putative transcription start sites
RNA PolII ChIP-Seq
Search for core promoter motifs

Additional TSS annotation resources

Structure of a typical mRNA

Pesole G. et al. Untranslated regions of mRNAs. Genome Biology. 2002: 3(3)

reviews0004.1-reviews0004.10.
 Muller F element, heterochromatic, and
euchromatic genes show similar expression levels

Riddle NC, et al. Enrichment of HP1a on Drosophila chromosome 4 genes creates an

alternate chromatin structure critical for regulation in this heterochromatic domain.
PLoS Genet. 2012 Sep;8(9):e1002954.
Annotate transcription start sites (TSS)

Research goal:
Identify motifs that enable Muller F element genes to
function within a heterochromatic environment

Annotation goals:
Define search regions enriched in regulatory motifs
Define precise location of TSS if possible
Define search regions where TSS could be found
Document the evidence used to support the TSS
annotations
 Estimated evolutionary distances with
respect to D. melanogaster
D. melanogaster
D. simulans Species Substitutions per
D. sechellia
D. yakuba
neutral site
D. erecta D. ficusphila 0.80
D. ficusphila
D. eugracilis D. eugracilis 0.76
D. biarmipes
D. takahashii D. biarmipes 0.70
D. elegans D. takahashii 0.65
D. rhopaloa
D. kikkawai
D. elegans 0.72
D. bipectinata D. rhopaloa 0.66
D. ananassae
D. pseudoobscura D. kikkawai 0.89
D. persimilis D. bipectinata 0.99
D. willistoni
Data from table 1 of the modENCODE
D. mojavensis comparative genomics whitepaper
D. virilis
D. grimshawi
New species sequenced by modENCODE GEP annotation projects
 TSS annotation resources
Walkthroughs:
Annotation of Transcription Start Sites in Drosophila

Reference:
TSS Annotation Workflow

GEP Annotation Report:

TSS Report Form (sample report for onecut)

Additional evidence tracks:

RNA PolII ChIP-Seq (D. biarmipes only)
D. mel Transcripts
Conservation (Multiz alignments)
 TSS annotation workflow
1. Identify the ortholog
2. Note the gene structure in D. melanogaster
3. Annotate the coding exons
4. Classify the type of core promoter in D. melanogaster
5. Annotate the initial transcribed exon
6. Identify any core promoter motifs
7. Define TSS positions or TSS search regions
 Evidence for TSS annotations
(in general order of importance)
1. Experimental data
RNA-Seq
RNA Pol II ChIP-Seq (D. biarmipes only)

2. Conservation
Type of TSS (peaked versus broad) in D. melanogaster
Sequence similarity to initial exon in D. melanogaster
Sequence similarity to other Drosophila species (Multiz)

3. Core promoter motifs

Inr, TATA box, etc.
Challenges with TSS annotations
Fewer constraints on untranslated regions (UTRs)
UTRs evolve more quickly than coding regions
Open reading frames, compatible phases of donor and
acceptor sites do not apply to UTRs

Low percent identity (~50-70%) between D. biarmipes

contigs and D. melanogaster UTRs
Most gene finders do not predict UTRs
Lack of experimental data
Cannot use RNA-Seq data to precisely define the TSS
 RNA-Seq biases introduced by
library construction

cDNA fragmentation
RNA-Seq Read Count

Strong bias at the 3’ end

RNA fragmentation
More uniform coverage
Miss the 5’ and 3’ ends
of the transcript

5’ 3’
Gene Span

Wang Z, et al. RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet. 2009 Jan;10(1):57-63.
 Techniques for finding TSS
Identify the 5’ cap at the beginning of the mRNA
Cap Analysis of Gene Expression (CAGE)
RNA Ligase Mediated Rapid Amplification of cDNA Ends
(RLM-RACE)
Cap-trapped Expressed Sequence Tags (5’ ESTs)

More information on these techniques:

Takahashi H, et al. CAGE (cap analysis of gene expression): a protocol
for the detection of promoter and transcriptional networks. Methods Mol
Biol. 2012 786:181-200.
Sandelin A, et al. Mammalian RNA polymerase II core promoters:
insights from genome-wide studies. Nat Rev Genet. 2007 Jun;8(6):424-36.
modENCODE TSS annotations
TSS predictions based on CAGE, RLM-RACE,
and 5’ EST data
Results lifted from Release 5 to the Release 6 assembly

Two sets of modENCODE TSS predictions

TSS (Celniker)
Most recent dataset produced by modENCODE
TSS (Embryonic)
Older dataset available from FlyBase GBrowse

Use TSS (Celniker) dataset as the primary evidence

Revised TSS analysis for Release 6 not yet available
Hoskins RA, et al. Genome-wide analysis of promoter architecture in Drosophila melanogaster.
Genome Res. 2011 Feb;21(2):182-92
 FlyBase release 6 assembly

First change in the D. melanogaster assembly since 2005

Substantial changes in the heterochromatic regions

modENCODE RNA-Seq data have been re-analyzed

relative to the release 6 assembly
Most of the modENCODE datasets are still in release 5
 Gnomon predictions for eight
Drosophila species
Based on RNA-Seq data from either the same or
closely-related species
D. simulans, D. yakuba, D. erecta, D. ananassae, D. pseudoobscura, D.
willistoni, D. virilis, and D. mojavensis
Predictions include untranslated regions and multiple isoforms
Records not yet available through the NCBI RefSeq database
 RNA Polymerase II core promoter

Initiator motif (Inr)

contains the TSS

TFIID binds to the

TATA box and Inr
to initiate the
assembly of the pre-
initiation complex
(PIC)

Juven-Gershon T and Kadonaga JT. Regulation of gene expression via the core promoter
and the basal transcriptional machinery. Dev Biol. 2010 Mar 15;339(2):225-9.
 Peaked versus broad promoters
Peaked promoter Broad promoter
(Single strong TSS) (Multiple weak TSS)

50-300 bp

Kadonaga JT. Perspectives on the RNA polymerase II core promoter. Wiley Interdiscip
Rev Dev Biol. 2012 Jan-Feb;1(1):40-51.
Promoter architecture in Drosophila
Classify core promoter based on
the Shape Index (SI)
Determined by distribution of
CAGE and 5’ RLM-RACE reads
Shape index is a continuum

Most promoters in D.
melanogaster contain multiple TSS
Median width = 162 bp

~70% of vertebrate genes have

broad promoters

Hoskins RA, et al. Genome-wide analysis of promoter

architecture in Drosophila melanogaster. Genome Res.
2011 Feb;21(2):182-92.
Genes with peaked promoters show stronger
spatial and tissue specificity

46% of genes with

broad promoters are
expressed in all
stages of embryonic
development

19% of genes with

peaked promoters
are expressed in all
stages

Hoskins RA, et al. Genome-wide

analysis of promoter architecture in
Drosophila melanogaster. Genome
Res. 2011 Feb;21(2):182-92.
Peaked and broad promoters are enriched in
different core promoter motifs

Rach EA, et al. Motif composition, conservation and condition-specificity of single and
alternative transcription start sites in the Drosophila genome. Genome Biol. 2009;10(7):R73.
Using modENCODE data to classify the
type of core promoter in D. melanogaster
Only a subset of the modENCODE data are
available through FlyBase
Evidence tracks on the D. melanogaster GEP UCSC
Genome Browser [July 2014 (BDGP R6) assembly]:
FlyBase gene annotations (release 6.06)
modENCODE TSS (Celniker) annotations
DNase I hypersensitive sites (DHS)
9-state and 16-state chromatin models
RNA Pol II and CBP ChIP-Seq
Transcription factor binding site (TFBS) HOT spots
9-state chromatin model

Kharchenko PV, et al.

Comprehensive analysis of
the chromatin landscape in
Drosophila melanogaster.
Nature. 2011 Mar
24;471(7339):480-5.
DNaseI Hypersensitive Sites (DHS)
correspond to accessible regions
Aasland R and Stewart
AF. Analysis of DNaseI
hypersensitive sites in
chromatin by cleavage
in permeabilized cells.
Methods Mol Biol.
1999;119:355-62.

Ho JW, et al.
Comparative analysis of
metazoan chromatin
organization. Nature.
2014 Aug
28;512(7515):449-52.
Classifying the D. melanogaster core
promoter for each unique TSS
Peaked
Single annotated TSS with a single DHS position

Intermediate
Single annotated TSS with multiple DHS positions within
a 300bp window

Broad
Multiple annotated TSS with multiple DHS positions
within a 300bp window

Insufficient evidence
No TSS annotations or DHS positions
DEMO
Classifying the core promoter of D. melanogaster Rad23
 Additional DHS data from different
stages of embryonic development

DHS data
produced by the
BDTNP project

Evidence tracks:
Detected DHS Positions
(Embryos)
DHS Read Density
(Embryos)

Thomas S, et al. Dynamic reprogramming of chromatin accessibility during Drosophila

embryo development. Genome Biol. 2011;12(5):R43.
 Determine the gene structure in
D. melanogaster
FlyBase: GBrowse UTR CDS

Gene Record Finder: Transcript Details

 Identify the initial transcribed
exon using NCBI blastn
Retrieve the sequences of the initial exons from the
Transcript Details tab of the Gene Record Finder
Use placement of the flanking exons to reduce the
size of the search region if possible
Increase sensitivity of nucleotide searches
Change Program Selection to blastn
Change Word size to 7
Change Match/Mismatch Scores to +1, -1
Change Gap Costs to Existence: 2, Extension: 1
Optimize alignment parameters based on
expected levels of conservation

Derive alignment scores

using information theory
Relative entropy of target and
background frequencies

Match +2, Mismatch -3

optimized for 90% identity
Match +1, Mismatch -1
optimized for 75% identity

States DJ, et al. Improved Sensitivity of Nucleic Acid Database Searches Using Application-
Specific Scoring Matrices. Methods 3:66-70.
DEMO
blastn search of the initial transcribed exon of Rad23 against D. biarmipes
contig19
 RNA PolII ChIP-Seq tracks
(D. biarmipes only)
Show regions that are enriched in RNA Polymerase
II compared to input DNA

Gene Models

RNA PolII Peaks

RNA PolII
Enrichment

RNA-Seq
 Use core promoter motifs to
support TSS annotations
Some sequence motifs are enriched in the region
(~300 bp) surrounding the TSS
Some motifs (e.g., Inr, TATA) are well-characterized
Other motifs are identified based on computational analysis

Presence of core promoter motifs can be used to

support the TSS annotations
Inr motif (TCAKTY) overlaps with the TSS (-2 to +4)

Absence of core promoter motifs is a negative result

Most D. melanogaster TSS do not contain the Inr motif
Use UCSC Genome Browser Short Match to
find Drosophila core promoter motifs
TATA box

Initiator (Inr)

Ohler U, et al. Computational

analysis of core promoters in
the Drosophila genome.
Genome Biol. 2002;
3(12):RESEARCH0087.

Available under “Projects”  “Annotation Resources”  “Core Promoter Motifs” on

the GEP web site: http://gander.wustl.edu/~wilson/core_promoter_motifs.html
Core Promoter Motifs tracks

Show core promoter motif matches for each contig

Separated by strand

Visualize matches to different core promoter motifs

Use UCSC Table Browser (or other means) to export
the list of motif matches within the search region
DEMO
Use the Inr motif to determine the TSS position of Rad23
Using RNA PolII ChIP-Seq and RNA-Seq
data to define the TSS search region

D. mel Transcripts

RNA PolII Peaks

RNA PolII
Enrichment

RNA-Seq

TSS search region

 TSS annotation for Rad23
TSS position: 28,936
Conservation with D. melanogaster
blastn of initial exon and the D. mel Transcripts track
Location of the Inr motif

TSS search region: 28,716-28,936

Enrichment of RNA PolII upstream of the TSS position
RNA-Seq read coverage upstream of the TSS position
Search region defined by the extent of the RNA PolII peak
TSS annotation section in the
GEP Annotation Report Form
TSS report form
Classify the type of core promoter
Evidence that supports or refutes the TSS annotation
Distribution of core promoter motifs in the project and
in D. melanogaster

Sample TSS report for onecut on the GEP web site

Under the “Beyond Annotation” section
 D. biarmipes Muller F element
TSS annotation projects
Reconcile coding region annotations submitted by
GEP students

Reconciled annotations might be incorrect

Based on older FlyBase release
Possible misannotations

See the “Revised gene models report form” section

of the Transcription Start Sites Project Report
Additional TSS annotation resources
The D. melanogaster gene annotations are the primary
source of evidence
Resources that might be useful if the D. melanogaster
evidence is ambiguous
Whole genome alignments of 14 Drosophila species
PhastCons and PhyloP conservation scores

Genome browsers for nine Drosophila species

RNA Pol II ChIP-Seq (D. biarmipes only)
RNA-Seq coverage, TopHat junctions, assembled transcripts
Augustus and N-SCAN gene predictions
Conservation tracks on the D. melanogaster
GEP UCSC Genome Browser
Whole genome alignments of 14 Drosophila species
Drosophila Chain/Net composite track

Generate multiple sequence (Multiz) alignments from

these pairwise alignments
Identify conserved regions from Multiz alignments
PhastCons: identify conserved elements
PhyloP: measure level of selection at each nucleotide

Multiz alignment of 27 insect species available on the

official UCSC Genome Browser
Aug. 2014 (BDGP Release 6 + ISO1 MT/dm6) assembly
Use the conservation tracks to identify
regions under selection

PhyloP scores: Under negative selection Fast-evolving

 Examine the Multiz alignments to
identify the orthologous TSS regions
Use RNA PolII tracks on the D. biarmipes
genome browser to identify putative TSS
April 2013 (BCM-HGSC/Dbia_2.0) assembly
Search for orthologous regions in D. elegans
 Use RNA-Seq data to predict untranslated
regions and putative TSS
TSS predictions available for 9 Drosophila species
N-SCAN+PASA-EST, Augustus, TransDecoder

D. mel
Proteins
N-SCAN
Augustus
TransDecoder

RNA-Seq
DEMO
Genome browsers for other Drosophila species
 TSS annotation summary
Most of the D. melanogaster core promoters have
multiple TSS
Classify the type of promoter (peaked/intermediate/broad)
based on the transcriptome evidence from D. melanogaster
Define search regions that might contain TSS

Use multiple lines of evidence to infer the TSS region

Identify initial exon
RNA-Seq coverage
blastn (change search parameters)
RNA PolII ChIP-Seq peaks
Distribution of core promoter motifs (e.g., Inr)
Maintain conservation compared to D. melanogaster
Questions?