Core promoter

1
Core Promoters Transcription Start Sites

• Core promoter
• region of 100 bp flanking the transcription
  start site
• recognition site for the basal transcription
  apparatus
• Implicated in gene regulation, not just
  initiation
• Identified sequence motifs
• Inr centered at position 1 25 of promoters
• TATA box centered at -40 20 of promoters
• DPE centered at 20 8 of promoters
• DRE DNA replication-related-element 25
• 6 more motifs defined computationally

2
Transcription Start Sites

• Full-Length cDNAs
• Cap-trapped libraries
• 5 ESTs directed cDNA screening
• 5 RACE primer extension
• Whole genome tiling arrays
• Tissue- and stage-specificity
• Alternative 5 exons
• Comparative genomics may help

3
Core Promoter Prediction Example of Ohler et al.
2002

• Selected 2,000 clusters of 5 ESTs
• 3 ESTs within 11 bp
• 1 cap-trapped EST
• 5-most EST defines position 1
• Find overrepresented words
• Search within -60 to 40
• MEME finds 10 sequence motifs 4 known and 6
  novel
• Re-train and run McPromoter
• Evaluate on Adh region and chromosome arm 2R
• Sensitivity / Specifity 65 / 29 to 19 / 69
  (threshold-dependent)
• Conclusion Good enough to direct wet lab
  experiments

4
P-elements Target Promoters

• gt 50,000 mapped insertions
• Insertions associated with 40 of genes

5
Drosophila Promoter Annotation
P-element insertion sites Promoter
predictions Curated gene models - open boxes
5UTRs
6
DNase I Hypersensitive Sites

• Sites of Accessible or Open Chromatin
• Southern Blot Assay Method
• Partial digestion of chromatin with DNase I
• Complete digestion with a restriction enzyme
• Southern blot to identify and locate site(s)
• DNase digest is partial, so fragments are
  size-fractionated prior to detection
• Related Methods
• Restriction enzyme hypersensitivity
• Chemical modification

7
Additional Methods

• ENCODE Methods
• ChIP-chip of proteins at promoters
• RNA pol II, TBP, etc.
• Transfection of reporter constructs
• DNase I hypersensitive sites
• EST-like sequencing of plasmid libraries
• Quantitative PCR detection
• Other Approaches
• In vitro transcription assays
• What else?

8
Sample ENCODE Region (Fig.3)

• Promoter finding methods correlate with each
  other and with 5 exons
• Functional assays of constructs look good
• RNA Pol II ChIP-chip can be very effective and
  scales to the genome
• DNaseI hypersenstitve sites correlate with
  promoters
• Some genes appear to have promoters at both ends
  antisense?

9
Summary of Methods

• Transcription Start Sites
• 5-ESTs, cDNA screening, and 5-RACE are scalable
• Whole genome tiling array hybridization is a key
• Core Promoters
• ChIP-chip with promoter-associated proteins is a
  key
• Reporter constructs tested in functional assays
  in fly cells?
• DNase hypersensitve sites
• Will high-throughput methods be effective and
  scalable?
• Are hypersenstive sites mostly promoters?
• Computational Methods
• Refinements to promoter prediction
• Comparative sequence analysis
• Comparative promoter prediction?

10
Promoter Annotation in Drosophila

• 14,000 protein-coding genes
• gt10,000 associated with 5 ESTs
• How many alternative promoters?
• Release 3 annotation suggests 3 of genes
• How many non-protein-coding genes?
• Project Scale 20,000 fly promoters?