De novo identification and biophysical characterization of transcription-factor binding sites with microfluidic affinity analysis

Abstract

Gene expression is regulated in part by protein transcription factors (TFs) that bind target regulatory DNA sequences. Predicting DNA binding sites and affinities from transcription factor sequence or structure is difficult; therefore, experimental data are required to link TFs to target sequences. We present a microfluidics-based approach for de novo discovery and quantitative biophysical characterization of DNA target sequences. We validated our technique by measuring sequence preferences for 28 S. cerevisiae TFs with a variety of DNA binding domains, including several that have proven difficult to study via other techniques. For each TF, we measured relative binding affinities to oligonucleotides covering all possible 8-bp DNA sequences to create a comprehensive map of sequence preferences; for 4 TFs, we also determined absolute affinities. We anticipate that these data and future use of this technique will provide information essential for understanding TF specificity, improving identification of regulatory sites, and reconstructing regulatory interactions.

Massively parallel measurements of molecular interaction kinetics on a microfluidic platform

“…1). MITOMI is a versatile platform capable of measuring a broad range of biomolecular interactions, including proteinprotein (27), protein-DNA (17,28), protein-RNA (29), and protein-small molecule (29). The integrated nature of the approach allows for the large-scale on-chip synthesis, purification, and characterization of proteins (30,31).…”

“…The topologies of these networks have been mapped with a variety of in vivo and in vitro methods, including ChIP-chip (7), ChIP-seq (5), Y1H (37), PBMs (38), SELEX (39,40), and MITOMI (17,28). The principal goals of these methods are to establish the genomic binding locations of TFs and to determine their consensus binding sequences, position weight matrixes, and binding energy landscapes (32, 41).…”

Basic leucine zipper transcription factor Hac1 binds DNA in two distinct modes as revealed by microfluidic analyses

“…1A). In previous work, we validated this platform using a panel of 28 S. cerevisiae transcription factors and demonstrated the ability to quantitatively measure relative binding affinities to each oligonucleotide and recover known binding preferences (18).…”

“…Experiments took place in six main steps ( surfaces beneath button valves that were coated with anti-His antibodies; (iii) protein solution was pushed into DNA compartments, solubilizing spotted DNA and allowing Hac1 i and DNA sequences to interact; (iv) binding interactions were mechanically trapped at equilibrium by pressurizing button valves to squeeze out unbound material; (v) neck valves were closed to isolate the compartments and allow washing away of unbound material in the protein compartment while preserving equilibrium concentrations of both binding partners in the DNA compartment; and (vi) devices were read using a fluorescence scanner. Final Cy5 intensities in each DNA chamber were previously shown to be proportional to the soluble DNA concentration available for binding (17,18), and the ratio of Cy5 (DNA) to BODIPY-FL (Hac1 i ) intensities beneath the button valve reports the protein fractional occupancy, allowing calculation of interaction K d and ΔΔG (Fig. 1C).…”

Budding off: bringing functional genomics toCandida albicans

“…A detailed analysis of this circuit included ChIP-chip, global transcriptome analysis and mechanically induced trapping of molecular interactions (MITOMI) [75]. The latter technique involves testing the binding of an in vitro translated protein to all possible 8-mer DNA sequences to determine DNA-binding specificity [79,80]. Using ChIP-chip, the six transcription factors regulating the white-opaque switch were found to bind to a restricted number of genomic regions (72-370), with significant overlap among the binding sites for all six proteins.…”