In vitro DNA-binding profile of transcription factors: methods and new insights

Abstract

The DNA-binding specificity of transcription factors (TFs) has broad impacts on cell physiology, cell development and in evolution. However, the DNA-binding specificity of most known TFs still remains unknown. The specificity of a TF protein is determined by its relative affinity to all possible binding sites. In recent years, the development of several in vitro techniques permits high-throughput determination of relative binding affinity of a TF to all possible k bp-long DNA sequences, thus greatly promoting the characterization of DNA-binding specificity of many known TFs. All DNA sequences that can be bound by a TF with various binding affinities form their DNA-binding profile (DBP). The DBP is important to generate an accurate DNA-binding model, identify all DNA-binding sites and target genes of TFs in the whole genome, and build transcription regulatory network. This study reviewed these techniques, especially two master techniques: double-stranded DNA microarray and systematic evolution of ligands by exponential enrichment in combination with parallel DNA sequencing techniques (SELEX-seq).

Defining the DNA uptake specificity of naturally competent Haemophilus influenzae cells

“…Position G5 is especially significant—it is not part of the inner core, but like those positions its information content has decreased relative to its A and T neighbours. The effects of base composition bias may have been further amplified by the bias towards short tracts of As, Ts and ATs that has been previously characterized in the H. influenzae genome, and these biases are even stronger in intergenic regions than in genes (56,57). …”

DNA‐Binding Small Molecules as Inhibitors of Transcription Factors

“…In recent years, several promising strategies for the identification of transcription factor binding site have been developed for the high‐throughput determination of transcription factor relative binding affinity to all possible sites in a long DNA sequence, such as double‐stranded DNA microarray and SELEX‐seq. Readers interested in the latest developments in analytical techniques for characterizing transcription factor DNA‐binding profiles are directed to a recent review by Liu and colleagues …”

The impact of intronic single nucleotide polymorphisms and ethnic diversity for studies on the obesity gene FTO

“…First of all, it requires antibodies with extremely high affinity and specificity for the target transcription factors to be successful in order to avoid cross‐reactivity of antibodies with closely related family members. Unfortunately, such antibodies are not available for most human transcription factors, producing non‐specific enrichment profiles (70). Second, as ChIP‐based methods only identify transcription binding factors in a particular cell at a specific time point, it is not possible to evaluate different conditions or developmental stages.…”