Combining SELEX with Combining SELEX with quantitative assays to rapidly quantitative assays to rapidly

obtain accurate models of obtain accurate models of protein–DNA interactionsprotein–DNA interactions

Jiajian Liu and Gary D. StormoJiajian Liu and Gary D. Stormo

Presented by Aliya SadequePresented by Aliya Sadeque

Protein-DNA interactionsProtein-DNA interactions

Methods for measuring:Methods for measuring:Yeast 1 hybridYeast 1 hybridChIP on chip, DNA microarrayChIP on chip, DNA microarray

Important distinction in terms of specificityImportant distinction in terms of specificityEnzymes vs. transcription factorsEnzymes vs. transcription factors

Bioinformatics. 2000 Jan;16(1):16-23.

TFBSTFBSTranscription Factor Binding SitesTranscription Factor Binding Sites

GoalGoal: Knowing the specificity of a TF in : Knowing the specificity of a TF in order to locate its binding sites within the order to locate its binding sites within the genome genome

Sites represented as consensus Sites represented as consensus sequences or weight matricessequences or weight matrices

Databases: TRANSFAC, JASPARDatabases: TRANSFAC, JASPAR

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SELEXSELEXSystematic Evolution of Ligands By Exponential EnrichmentSystematic Evolution of Ligands By Exponential Enrichment

http://rulai.cshl.edu/tools/ESE2/ESEmatrix.html

QuMFRAQuMFRA

Nucleic Acids Res. 2001 Jun 15;29(12):2471-8. PMID: 11410653

Selling pointsSelling points

Provides a general Provides a general method that can be method that can be used for any DNA-used for any DNA-binding protein even if binding protein even if nothing is known nothing is known about its specificity. about its specificity.

Can isolate a small Can isolate a small set of specific binding set of specific binding sites from a very large sites from a very large pool of random pool of random sequencessequences

Nucleic Acids Res. 2001 Jun 15;29(12):2471-8.

Zif268Zif268

SELEX ProcedureSELEX Procedure

SELEX Binding ModelSELEX Binding Model

Assuming an additive modelAssuming an additive model Frequency:Frequency:

WeightWeight

Sequence LogoSequence Logoobtained from SELEXobtained from SELEX

QuMFRA ProcedureQuMFRA Procedure

15 sequences15 sequencesCover the space of possible sequenceCover the space of possible sequenceCompetitive binding assayCompetitive binding assay

http://www.answers.com/topic/gel-abpp-eg-png

Uh oh, math.Uh oh, math.

Intensities of each DNAIntensities of each DNA in a separated band in a separated band

Obtained from Obtained from emission matrixemission matrix and and output vectoroutput vector

Relative binding constant of a test site with Relative binding constant of a test site with respect to a reference site respect to a reference site Reference was GGGTReference was GGGT

bound unbound

QuMFRA Binding ModelQuMFRA Binding Model

Weight matrix Consensus Sequence

Matrix values are proportional to binding affinity according to the Berg and von Hippel theory.

Comparing notesComparing notesBetween SELEX and QuMFRABetween SELEX and QuMFRA

Experimental Experimental KKaafor 15 sequencesfor 15 sequences

RenormalizedRenormalizedon consensuson consensussequencesequence

Comparing notesComparing notesBetween predictions and empirical binding affinitiesBetween predictions and empirical binding affinities

Found affinity measures for 8 variants of Found affinity measures for 8 variants of the consensus sequence with one or two the consensus sequence with one or two changed positionschanged positions

A Probabilitistic Recognition CodeA Probabilitistic Recognition CodeI promise those are all real wordsI promise those are all real words

AlternativesAlternatives SAGE-SELEXSAGE-SELEX

Improves on the Improves on the number of binding number of binding sites found by SELEX sites found by SELEX alonealone

Large sample size Large sample size reqired for statistical reqired for statistical significancesignificance

Biases in SELEXBiases in SELEX

AlternativesAlternatives

dsDNA chips dsDNA chips Chips contain binding Chips contain binding

sites for a TF of sites for a TF of interestinterest

High throughput High throughput quantitative dataquantitative data

Almost all possible Almost all possible binding sites would binding sites would have to be on the have to be on the chip…that’s a lot.chip…that’s a lot.

AlternativesAlternatives

Similar SequencesSimilar Sequences optimized selection of DNA variants to be optimized selection of DNA variants to be

tested experimentallytested experimentally quantitative protein-DNA binding assayquantitative protein-DNA binding assay prediction of binding affinity for all variants prediction of binding affinity for all variants

using a statistical modelusing a statistical model

Can be done in high throughput with high Can be done in high throughput with high accuracy, provided the accuracy, provided the consensus consensus sequence is knownsequence is known

PraisesPraises

General:General:Can be used for a TF of unknown specificity Can be used for a TF of unknown specificity

and and sizesizeEfficientEfficient

Parallel (different colours of fluorophore)Parallel (different colours of fluorophore)First step narrows down sample sizeFirst step narrows down sample size

HecklesHeckles

Sequenced a sample of ~20 - too small.Sequenced a sample of ~20 - too small.Could skew data. Secondary preference?Could skew data. Secondary preference?

Why no A at position 1 in QuMFRA?Why no A at position 1 in QuMFRA?Weakness/inherent bias in SELEXWeakness/inherent bias in SELEXEmpirical data - is an average Empirical data - is an average

appropriate?appropriate?

HecklesHeckles

Correlation values (0.54, 0.6, 0.77, 0.94) – Correlation values (0.54, 0.6, 0.77, 0.94) – are these significant? No T-test or are these significant? No T-test or anything?anything?

Questions?Questions?