Combining SELEX with quantitative assays to rapidly obtain accurate models of protein–DNA interactions Jiajian Liu and Gary D. Stormo Presented by Aliya Sadeque

Protein-DNA interactions Methods for measuring: Methods for measuring: Yeast 1 hybrid Yeast 1 hybrid ChIP on chip, DNA microarray ChIP on chip, DNA microarray Important distinction in terms of specificity Important distinction in terms of specificity Enzymes vs. transcription factors Enzymes vs. transcription factors Bioinformatics Jan;16(1):16-23.

TFBS Transcription Factor Binding Sites Goal: Knowing the specificity of a TF in order to locate its binding sites within the genome Goal: Knowing the specificity of a TF in order to locate its binding sites within the genome Sites represented as consensus sequences or weight matrices Sites represented as consensus sequences or weight matrices Databases: TRANSFAC, JASPAR Databases: TRANSFAC, JASPAR

SELEX Systematic Evolution of Ligands By Exponential Enrichment

QuMFRA Nucleic Acids Res Jun 15;29(12): PMID:

Selling points Provides a general method that can be used for any DNA- binding protein even if nothing is known about its specificity. Provides a general method that can be used for any DNA- binding protein even if nothing is known about its specificity. Can isolate a small set of specific binding sites from a very large pool of random sequences Can isolate a small set of specific binding sites from a very large pool of random sequences Nucleic Acids Res Jun 15;29(12):

Zif268

SELEX Procedure

SELEX Binding Model Assuming an additive model Assuming an additive model Frequency: Frequency: Weight Weight

Sequence Logo obtained from SELEX

QuMFRA Procedure 15 sequences 15 sequences Cover the space of possible sequence Cover the space of possible sequence Competitive binding assay Competitive binding assay

Uh oh, math.  Intensities of each DNA in a separated band Intensities of each DNA in a separated band Obtained from emission matrix and output vector Obtained from emission matrix and output vector Relative binding constant of a test site with respect to a reference site Relative binding constant of a test site with respect to a reference site Reference was GGGT Reference was GGGT boundunbound

QuMFRA Binding Model Weight matrixConsensus Sequence Matrix values are proportional to binding affinity according to the Berg and von Hippel theory.

Comparing notes Between SELEX and QuMFRA Experimental K a for 15 sequences Experimental K a for 15 sequences Renormalized on consensus sequence Renormalized on consensus sequence

Comparing notes Between predictions and empirical binding affinities Found affinity measures for 8 variants of the consensus sequence with one or two changed positions Found affinity measures for 8 variants of the consensus sequence with one or two changed positions

A Probabilitistic Recognition Code I promise those are all real words

Alternatives SAGE-SELEX Improves on the number of binding sites found by SELEX alone Improves on the number of binding sites found by SELEX alone Large sample size reqired for statistical significance Large sample size reqired for statistical significance Biases in SELEX Biases in SELEX

Alternatives dsDNA chips Chips contain binding sites for a TF of interest Chips contain binding sites for a TF of interest High throughput quantitative data High throughput quantitative data Almost all possible binding sites would have to be on the chip…that’s a lot. Almost all possible binding sites would have to be on the chip…that’s a lot.

Alternatives Similar Sequences optimized selection of DNA variants to be tested experimentally optimized selection of DNA variants to be tested experimentally quantitative protein-DNA binding assay quantitative protein-DNA binding assay prediction of binding affinity for all variants using a statistical model prediction of binding affinity for all variants using a statistical model Can be done in high throughput with high accuracy, provided the consensus sequence is known Can be done in high throughput with high accuracy, provided the consensus sequence is known

Praises General: Can be used for a TF of unknown specificity and size Can be used for a TF of unknown specificity and size Efficient Efficient Parallel (different colours of fluorophore) Parallel (different colours of fluorophore) First step narrows down sample size First step narrows down sample size

Heckles 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 SELEX Weakness/inherent bias in SELEX Empirical data - is an average appropriate? Empirical data - is an average appropriate?

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

Questions?