Transcription factors (TFs) recognize specific DNA sequences to control chromatin and transcription, forming a complex system that guides expression of the genome. Despite keen interest in understanding how TFs control gene expression, it remains challenging to determine how the precise genomic binding sites of TFs are specified and how TF binding ultimately relates to regulation of transcription. This review considers how TFs are identified and functionally characterized, principally through the lens of a catalog of over 1,600 likely human TFs and binding motifs for two-thirds of them. Major classes of human TFs differ markedly in their evolutionary trajectories and expression patterns, underscoring distinct functions. TFs likewise underlie many different aspects of human physiology, disease, and variation, highlighting the importance of continued effort to understand TF-mediated gene regulation.

The majority of CpG dinucleotides in the human genome are methylated at cytosine bases. However, active gene regulatory elements are generally hypomethylated relative to their flanking regions, and the binding of some transcription factors (TFs) is diminished by methylation of their target sequences. By analysis of 542 human TFs with methylation-sensitive SELEX (systematic evolution of ligands by exponential enrichment), we found that there are also many TFs that prefer CpG-methylated sequences. Most of these are in the extended homeodomain family. Structural analysis showed that homeodomain specificity for methylcytosine depends on direct hydrophobic interactions with the methylcytosine 5-methyl group. This study provides a systematic examination of the effect of an epigenetic DNA modification on human TF binding specificity and reveals that many developmentally important proteins display preference for mCpG-containing sequences.

Gene expression is regulated by transcription factors (TFs), proteins that recognize short DNA sequence motifs. Such sequences are very common in the human genome, and an important determinant of the specificity of gene expression is the cooperative binding of multiple TFs to closely located motifs. However, interactions between DNA-bound TFs have not been systematically characterized. To identify TF pairs that bind cooperatively to DNA, and to characterize their spacing and orientation preferences, we have performed consecutive affinity-purification systematic evolution of ligands by exponential enrichment (CAP-SELEX) analysis of 9,400 TF-TF-DNA interactions. This analysis revealed 315 TF-TF interactions recognizing 618 heterodimeric motifs, most of which have not been previously described. The observed cooperativity occurred promiscuously between TFs from diverse structural families. Structural analysis of the TF pairs, including a novel crystal structure of MEIS1 and DLX3 bound to their identified recognition site, revealed that the interactions between the TFs were predominantly mediated by DNA. Most TF pair sites identified involved a large overlap between individual TF recognition motifs, and resulted in recognition of composite sites that were markedly different from the individual TF's motifs. Together, our results indicate that the DNA molecule commonly plays an active role in cooperative interactions that define the gene regulatory lexicon.

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Divergent morphology of species has largely been ascribed to genetic differences in the tissue-specific expression of proteins, which could be achieved by divergence in cis-regulatory elements or by altering the binding specificity of transcription factors (TFs). The relative importance of the latter has been difficult to assess, as previous systematic analyses of TF binding specificity have been performed using different methods in different species. To address this, we determined the binding specificities of 242 Drosophila TFs, and compared them to human and mouse data. This analysis revealed that TF binding specificities are highly conserved between Drosophila and mammals, and that for orthologous TFs, the similarity extends even to the level of very subtle dinucleotide binding preferences. The few human TFs with divergent specificities function in cell types not found in fruit flies, suggesting that evolution of TF specificities contributes to emergence of novel types of differentiated cells.DOI: http://dx.doi.org/10.7554/eLife.04837.001

Nucleosomes cover most of the genome and are thought to be displaced by transcription factors (TFs) in regions that direct gene expression. However, the modes of interaction between TFs and nucleosomal DNA remain largely unknown. Here, we have systematically explored interactions between the nucleosome and 220 TFs representing diverse structural families. Consistently with earlier observations, we find that the majority of the studied TFs have less access to nucleosomal DNA than to free DNA. The motifs recovered from TFs bound to nucleosomal and free DNA are generally similar; however, steric hindrance and scaffolding by the nucleosome result in specific positioning and orientation of the motifs. Many TFs preferentially bind close to the end of nucleosomal DNA, or to periodic positions at its solvent-exposed side. TFs often also bind to nucleosomal DNA in a particular orientation. Some TFs specifically interact with DNA located at the dyad position where only one DNA gyre is wound, whereas other TFs prefer sites spanning two DNA gyres and bind specifically to each of them. Our work reveals striking differences in TF binding to free and nucleosomal DNA, and uncovers a rich interaction landscape between TFs and the nucleosome.

Transcription factors (TFs) achieve DNA‐binding specificity through contacts with functional groups of bases (base readout) and readout of structural properties of the double helix (shape readout). Currently, it remains unclear whether DNA shape readout is utilized by only a few selected TF families, or whether this mechanism is used extensively by most TF families. We resequenced data from previously published HT‐SELEX experiments, the most extensive mammalian TF–DNA binding data available to date. Using these data, we demonstrated the contributions of DNA shape readout across diverse TF families and its importance in core motif‐flanking regions. Statistical machine‐learning models combined with feature‐selection techniques helped to reveal the nucleotide position‐dependent DNA shape readout in TF‐binding sites and the TF family‐specific position dependence. Based on these results, we proposed novel DNA shape logos to visualize the DNA shape preferences of TFs. Overall, this work suggests a way of obtaining mechanistic insights into TF–DNA binding without relying on experimentally solved all‐atom structures.

C2H2 zinc finger proteins represent the largest and most enigmatic class of human transcription factors. Their C2H2-ZF arrays are highly variable, indicating that most will have unique DNA binding motifs. However, most of the binding motifs have not been directly determined. In addition, little is known about whether or how these proteins regulate transcription. Most of the ∼700 human C2H2-ZF proteins also contain at least one KRAB, SCAN, BTB, or SET domain, suggesting that they may have common interacting partners and/or effector functions. Here, we report a multifaceted functional analysis of 131 human C2H2-ZF proteins, encompassing DNA binding sites, interacting proteins, and transcriptional response to genetic perturbation. We confirm the expected diversity in DNA binding motifs and genomic binding sites, and provide motif models for 78 previously uncharacterized C2H2-ZF proteins, most of which are unique. Surprisingly, the diversity in protein–protein interactions is nearly as high as diversity in DNA binding motifs: Most C2H2-ZF proteins interact with a unique spectrum of co-activators and co-repressors. Thus, multiparameter diversification likely underlies the evolutionary success of this large class of human proteins.