1. Searching for Transcription Start Sites in Drosophila
Wilson Leung 08/2015

2. 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

3. Structure of a typical mRNA
Pesole G. et al. Untranslated regions of mRNAs. Genome Biology. 2002: 3(3) reviews reviews

4. Muller F element, heterochromatic, and euchromatic genes show similar expression levels
F element genes show lower expression levels in S2 (late embryos) cells but no difference in BG3 (neuronal) cells
See Dr. Elgin’s presentation on the private wiki for a more detailed explanation of the scientific goals.
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 Sep;8(9):e

5. 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
Detailed documentations allow us to prioritize the list of TSS candidates
In the process of analyzing the GEP submissions from this past year

6. Estimated evolutionary distances with respect to D. melanogaster
D. simulans
Species
Substitutions per neutral site
D. ficusphila
0.80
D. eugracilis
0.76
D. biarmipes
0.70
D. takahashii
0.65
D. elegans
0.72
D. rhopaloa
0.66
D. kikkawai
0.89
D. bipectinata
0.99
D. sechellia
D. yakuba
D. erecta
D. ficusphila
D. eugracilis
D. biarmipes
D. takahashii
D. elegans
D. rhopaloa
D. kikkawai
D. bipectinata
Purple = priority 1 species in modENCODE white paper
D. melanogaster subgroup = 0.4ss
D. ananassae = 1.3ss
D. ananassae
D. pseudoobscura
D. persimilis
D. willistoni
Data from table 1 of the modENCODE comparative genomics whitepaper
D. mojavensis
D. virilis
D. grimshawi
GEP annotation projects
New species sequenced by modENCODE

7. 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)
Additional curriculum on motif finding also available under the “Beyond annotation” section
Added new parameters page to the TSS Annotation Workflow

8. TSS annotation workflow
Identify the ortholog
Note the gene structure in D. melanogaster
Annotate the coding exons
Classify the type of core promoter in D. melanogaster
Annotate the initial transcribed exon
Identify any core promoter motifs
Define TSS positions or TSS search regions
Annotation is based on parsimony with D. melanogaster

9. Evidence for TSS annotations (in general order of importance)
Experimental data
RNA-Seq
RNA Pol II ChIP-Seq (D. biarmipes only)
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)
Core promoter motifs
Inr, TATA box, etc.

10. 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

11. RNA-Seq biases introduced by library construction
cDNA fragmentation
Strong bias at the 3’ end
RNA fragmentation
More uniform coverage
Miss the 5’ and 3’ ends of the transcript
RNA-Seq Read Count
This chart uses yeast (single ORF) as an example
It does not account for biases in read mapping
RNA fragmentation (e.g., hydrolysis, nebulization) missed the transcript ends
cDNA fragmentation (e.g., sonication, DNaseI) has strong bias at the 3’ end
Transcripts show more uniform coverage using RNA instead of cDNA fragmentation 5’ 3’
Gene Span
Wang Z, et al. RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet Jan;10(1):57-63.

12. 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 :
Sandelin A, et al. Mammalian RNA polymerase II core promoters: insights from genome-wide studies. Nat Rev Genet Jun;8(6):
CAGE randomly sample 5’ end – correspond to expression level
RACE target 5’ ends of specific transcripts (8727 transcripts) – capture lowly expressed transcripts
5’ end of mRNA has a guanine connected to the rest of the mRNA via a 5’ to 5’ triphosphate link.
This guanosine is methylated at position 7 (7-methylguanylate cap) and helps increase the stability of the mRNA.

13. 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
BDGP working with FlyBase to update the TSS annotations
Just updated RNA-Seq datasets in release 6.03
Hoskins RA, et al. Genome-wide analysis of promoter architecture in Drosophila melanogaster. Genome Res Feb;21(2):182-92

14. 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

15. 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
Access these annotations through the FlyBase BLAST service

16. 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)
polypyrimidine initiator (TCT) motif associated with ribosomal genes
Core promoter 200bp surrounding the TSS
K = G or T, W = A or T
Mammalian genes have CpG islands but Drosophila promoters do not
Juven-Gershon T and Kadonaga JT. Regulation of gene expression via the core promoter and the basal transcriptional machinery. Dev Biol Mar 15;339(2):225-9.

17. Peaked versus broad promoters
Peaked promoter
(Single strong TSS)
Broad promoter
(Multiple weak TSS) bp
Peaked promoter in D. melanogaster is more informative: expect peak promoter in the target species
focused (peaked) versus dispersed (broad) promoter
Motifs associated with peaked promoters have more well-defined positions relative to +1
Kadonaga JT. Perspectives on the RNA polymerase II core promoter. Wiley Interdiscip Rev Dev Biol Jan-Feb;1(1):40-51.

18. 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
median width 162bp => approximately the length of DNA for a single nucleosome
Most of the promoters in Drosophila are broad promoters
Motif associated with Paused polymerase are found in peaked promoters
Hoskins RA, et al. Genome-wide analysis of promoter architecture in Drosophila melanogaster. Genome Res Feb;21(2):

19. 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
Peaked promoter – have more precise control
Hoskins RA, et al. Genome-wide analysis of promoter architecture in Drosophila melanogaster. Genome Res Feb;21(2):

20. 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.

21. Using modENCODE data to classify the type of core promoter in D
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
16-state hiHMM models: late embryos and third instar larvae
9-state chromHMM models: S2 and BG3 cells
CREB-binding proteins – coactivation of transcription factors
DHS data available for different embryonic stages and cell lines

22. 9-state chromatin model
Kharchenko PV, et al. Comprehensive analysis of the chromatin landscape in Drosophila melanogaster. Nature Mar 24;471(7339):480-5.

23. 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:
Ho JW, et al. Comparative analysis of metazoan chromatin organization. Nature Aug 28;512(7515):

24. 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
Classification is based on each unique TSS
Different unique TSS for the same gene could have different classifications

25. DEMO
Classifying the core promoter of D. melanogaster Rad23

26. 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)
BDTNP = Berkeley Drosophila Transcription Network Project
Use DHS data from cell lines as primary evidence
Thomas S, et al. Dynamic reprogramming of chromatin accessibility during Drosophila embryo development. Genome Biol. 2011;12(5):R43.

27. Determine the gene structure in D. melanogaster
FlyBase: GBrowse
UTR
CDS
Gene Record Finder: Transcript Details

28. 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

29. 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
Less information available per aligned position
WU-BLAST: +5/-4, +5,-11
information = decrease in uncertainty
Convey more information from larger vocabulary and surprising answer (inversely proportional to its probability)
PAM = Point Accepted Mutations
Need aligned base to have some minimum information content to get significant alignment
Shannon Entropy = unpredictability of random variable
= subtract Kullback-Leibler divergence (target vs. uniform distribution) from total entropy required to encode a message
States DJ, et al. Improved Sensitivity of Nucleic Acid Database Searches Using Application-Specific Scoring Matrices. Methods 3:66-70.

30. DEMO
blastn search of the initial transcribed exon of Rad23 against D. biarmipes contig19

31. 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

32. 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
TCA for broad promoters

33. Use UCSC Genome Browser Short Match to find Drosophila core promoter motifs
Ohler U, et al. Computational analysis of core promoters in the Drosophila genome. Genome Biol. 2002; 3(12):RESEARCH0087.
TATA box
Initiator (Inr)
Available under “Projects”  “Annotation Resources”  “Core Promoter Motifs” on the GEP web site:

34. 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

35. DEMO
Use the Inr motif to determine the TSS position of Rad23

36. 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

37. 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

38. 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

39. 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
Example: some coding exon annotations are based on weak evidence (e.g., Small Exon Finder)
CDS might overlap with untranslated exons or TSS of an adjacent gene

40. 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
onecut example: supported by both RNA-Seq and Multiz

41. 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 (BDGP Release 6 + ISO1 MT/dm6) assembly

42. Use the conservation tracks to identify regions under selection
PhyloP scores:
Under negative selection
Fast-evolving

43. Examine the Multiz alignments to identify the orthologous TSS regions

44. Use RNA PolII tracks on the D
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 more stringent parameters than the GEP annotation projects (problem with overlapping projects).

45. 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

46. DEMO
Genome browsers for other Drosophila species

47. 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

48. Questions?