Hidden modes of DNA binding by human nuclear receptors.

Human nuclear receptors (NRs) are a superfamily of ligand-responsive transcription factors that have central roles in cellular function. Their malfunction is linked to numerous diseases, and the ability to modulate their activity with synthetic ligands has yielded 16% of all FDA-approved drugs. NRs regulate distinct gene networks, however they often function from genomic sites that lack known binding motifs. Here, to annotate genomic binding sites of known and unexamined NRs more accurately, we use high-throughput SELEX to comprehensively map DNA binding site preferences of all full-length human NRs, in complex with their ligands. Furthermore, to identify non-obvious binding sites buried in DNA-protein interactomes, we develop MinSeq Find, a search algorithm based on the MinTerm concept from electrical engineering and digital systems design. The resulting MinTerm sequence set (MinSeqs) reveal a constellation of binding sites that more effectively annotate NR-binding profiles in cells. MinSeqs also unmask binding sites created or disrupted by 52,106 single-nucleotide polymorphisms associated with human diseases. By implicating druggable NRs as hidden drivers of multiple human diseases, our results not only reveal new biological roles of NRs, but they also provide a resource for drug-repurposing and precision medicine.

Targeting an evolutionarily conserved "E-L-L" motif in the spike protein to develop a small molecule fusion inhibitor against SARS-CoV-2.

As newer variants of SARS-CoV-2 continue to pose major threats to global human health and economy, identifying novel druggable antiviral targets is the key towards sustenance. Here, we identify an evolutionary conserved E-L-L motif present within the HR2 domain of all human and non-human coronavirus spike (S) proteins that play a crucial role in stabilizing the post-fusion six-helix bundle (6-HB) structure and thus, fusion-mediated viral entry. Mutations within this motif reduce the fusogenicity of the S protein without affecting its stability or membrane localization. We found that posaconazole, an FDA-approved drug, binds to this E-L-L motif resulting in effective inhibition of SARS-CoV-2 infection in cells. While posaconazole exhibits high efficacy towards blocking S protein-mediated viral entry, mutations within the E-L-L motif rendered the protein completely resistant to the drug, establishing its specificity towards this motif. Our data demonstrate that posaconazole restricts early stages of infection through specific inhibition of membrane fusion and viral genome release into the host cell and is equally effective towards all major variants of concerns of SARS-CoV-2 including beta, kappa, delta, and omicron. Together, we show that this conserved essential E-L-L motif is an ideal target for the development of prophylactic and therapeutic interventions against SARS-CoV-2.

Targeting an evolutionarily conserved "E-L-L" motif in spike protein to identify a small molecule fusion inhibitor against SARS-CoV-2.

As newer variants of SARS-CoV-2 continue to pose major threats to global human health and economy, identifying novel druggable antiviral targets is the key toward sustenance. Here, we identify an evolutionarily conserved "Ex3Lx6L" ("E-L-L") motif present within the HR2 domain of all human and nonhuman coronavirus spike (S) proteins that play a crucial role in stabilizing its postfusion six-helix bundle (6-HB) structure and thus, fusion-mediated viral entry. Mutations within this motif reduce the fusogenicity of the S protein without affecting its stability or membrane localization. We found that posaconazole, an FDA-approved drug, binds to this "E-L-L" motif and impedes the formation of 6-HB, thus effectively inhibiting SARS-CoV-2 infection in cells. While posaconazole exhibits high efficacy in blocking S protein-mediated viral entry, mutations within the "E-L-L" motif rendered the protein completely resistant to the drug, establishing its specificity toward this motif. Our data demonstrate that posaconazole restricts early stages of infection through specific inhibition of membrane fusion and viral genome release into the host cell and is equally effective toward all major variants of concerns of SARS-CoV-2, including Beta, Kappa, Delta, and Omicron. Together, we show that this conserved essential "E-L-L" motif is an ideal target for the development of prophylactic and therapeutic interventions against SARS-CoV-2.

Single position substitution of hairpin pyrrole-imidazole polyamides imparts distinct DNA-binding profiles across the human genome.

Pyrrole-imidazole (Py-Im) polyamides are synthetic molecules that can be rationally designed to target specific DNA sequences to both disrupt and recruit transcriptional machinery. While in vitro binding has been extensively studied, in vivo effects are often difficult to predict using current models of DNA binding. Determining the impact of genomic architecture and the local chromatin landscape on polyamide-DNA sequence specificity remains an unresolved question that impedes their effective deployment in vivo. In this report we identified polyamide-DNA interaction sites across the entire genome, by covalently crosslinking and capturing these events in the nuclei of human LNCaP cells. This technique confirms the ability of two eight ring hairpin-polyamides, with similar architectures but differing at a single ring position (Py to Im), to retain in vitro specificities and display distinct genome-wide binding profiles.

De novo design of programmable inducible promoters.

Ligand-responsive allosteric transcription factors (aTF) play a vital role in genetic circuits and high-throughput screening because they transduce biochemical signals into gene expression changes. Programmable control of gene expression from aTF-regulated promoter is important because different downstream effector genes function optimally at different expression levels. However, tuning gene expression of native promoters is difficult due to complex layers of homeostatic regulation encoded within them. We engineered synthetic promoters de novo by embedding operator sites with varying affinities and radically reshaped binding preferences within a minimal, constitutive Escherichia coli promoter. Multiplexed cell-based screening of promoters for three TetR-like aTFs generated with this approach gave rich diversity of gene expression levels, dynamic ranges and ligand sensitivities and were 50- to 100-fold more active over their respective native promoters. Machine learning on our dataset revealed that relative position of the core motif and bases flanking the core motif play an important role in modulating induction response. Our generalized approach yields customizable and programmable aTF-regulated promoters for engineering cellular pathways and enables the discovery of new small molecule biosensors.

Flexibility and structure of flanking DNA impact transcription factor affinity for its core motif.

Spatial and temporal expression of genes is essential for maintaining phenotype integrity. Transcription factors (TFs) modulate expression patterns by binding to specific DNA sequences in the genome. Along with the core binding motif, the flanking sequence context can play a role in DNA-TF recognition. Here, we employ high-throughput in vitro and in silico analyses to understand the influence of sequences flanking the cognate sites in binding of three most prevalent eukaryotic TF families (zinc finger, homeodomain and bZIP). In vitro binding preferences of each TF toward the entire DNA sequence space were correlated with a wide range of DNA structural parameters, including DNA flexibility. Results demonstrate that conformational plasticity of flanking regions modulates binding affinity of certain TF families. DNA duplex stability and minor groove width also play an important role in DNA-TF recognition but differ in how exactly they influence the binding in each specific case. Our analyses further reveal that the structural features of preferred flanking sequences are not universal, as similar DNA-binding folds can employ distinct DNA recognition modes.

Specificity landscapes unmask submaximal binding site preferences of transcription factors.

We have developed Differential Specificity and Energy Landscape (DiSEL) analysis to comprehensively compare DNA-protein interactomes (DPIs) obtained by high-throughput experimental platforms and cutting edge computational methods. While high-affinity DNA binding sites are identified by most methods, DiSEL uncovered nuanced sequence preferences displayed by homologous transcription factors. Pairwise analysis of 726 DPIs uncovered homolog-specific differences at moderate- to low-affinity binding sites (submaximal sites). DiSEL analysis of variants of 41 transcription factors revealed that many disease-causing mutations result in allele-specific changes in binding site preferences. We focused on a set of highly homologous factors that have different biological roles but "read" DNA using identical amino acid side chains. Rather than direct readout, our results indicate that DNA noncontacting side chains allosterically contribute to sculpt distinct sequence preferences among closely related members of transcription factor families.

Combinatorial bZIP dimers display complex DNA-binding specificity landscapes.

How transcription factor dimerization impacts DNA-binding specificity is poorly understood. Guided by protein dimerization properties, we examined DNA binding specificities of 270 human bZIP pairs. DNA interactomes of 80 heterodimers and 22 homodimers revealed that 72% of heterodimer motifs correspond to conjoined half-sites preferred by partnering monomers. Remarkably, the remaining motifs are composed of variably-spaced half-sites (12%) or 'emergent' sites (16%) that cannot be readily inferred from half-site preferences of partnering monomers. These binding sites were biochemically validated by EMSA-FRET analysis and validated in vivo by ChIP-seq data from human cell lines. Focusing on ATF3, we observed distinct cognate site preferences conferred by different bZIP partners, and demonstrated that genome-wide binding of ATF3 is best explained by considering many dimers in which it participates. Importantly, our compendium of bZIP-DNA interactomes predicted bZIP binding to 156 disease associated SNPs, of which only 20 were previously annotated with known bZIP motifs.

Reprogramming cell fate with a genome-scale library of artificial transcription factors.

Artificial transcription factors (ATFs) are precision-tailored molecules designed to bind DNA and regulate transcription in a preprogrammed manner. Libraries of ATFs enable the high-throughput screening of gene networks that trigger cell fate decisions or phenotypic changes. We developed a genome-scale library of ATFs that display an engineered interaction domain (ID) to enable cooperative assembly and synergistic gene expression at targeted sites. We used this ATF library to screen for key regulators of the pluripotency network and discovered three combinations of ATFs capable of inducing pluripotency without exogenous expression of Oct4 (POU domain, class 5, TF 1). Cognate site identification, global transcriptional profiling, and identification of ATF binding sites reveal that the ATFs do not directly target Oct4; instead, they target distinct nodes that converge to stimulate the endogenous pluripotency network. This forward genetic approach enables cell type conversions without a priori knowledge of potential key regulators and reveals unanticipated gene network dynamics that drive cell fate choices.

Synthetic genome readers target clustered binding sites across diverse chromatin states.

Targeting the genome with sequence-specific DNA-binding molecules is a major goal at the interface of chemistry, biology, and precision medicine. Polyamides, composed of N-methylpyrrole and N-methylimidazole monomers, are a class of synthetic molecules that can be rationally designed to "read" specific DNA sequences. However, the impact of different chromatin states on polyamide binding in live cells remains an unresolved question that impedes their deployment in vivo. Here, we use cross-linking of small molecules to isolate chromatin coupled to sequencing to map the binding of two bioactive and structurally distinct polyamides to genomes directly within live H1 human embryonic stem cells. This genome-wide view from live cells reveals that polyamide-based synthetic genome readers bind cognate sites that span a range of binding affinities. Polyamides can access cognate sites within repressive heterochromatin. The occupancy patterns suggest that polyamides could be harnessed to target loci within regions of the genome that are inaccessible to other DNA-targeting molecules.

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC (Crosslinking of Small Molecules to Isolate Chromatin).

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to dose-limiting toxicity and many adverse side effects. Targeting the genome with sequence-specific small molecules may enable molecules with increased therapeutic index and fewer off-target effects. N-methylpyrrole/N-methylimidazole polyamides are molecules that can be rationally designed to target specific DNA sequences with exquisite precision. And unlike most natural transcription factors, polyamides can bind to methylated and chromatinized DNA without a loss in affinity. The sequence specificity of polyamides has been extensively studied in vitro with cognate site identification (CSI) and with traditional biochemical and biophysical approaches, but the study of polyamide binding to genomic targets in cells remains elusive. Here we report a method, the crosslinking of small molecules to isolate chromatin (COSMIC), that identifies polyamide binding sites across the genome. COSMIC is similar to chromatin immunoprecipitation (ChIP), but differs in two important ways: (1) a photocrosslinker is employed to enable selective, temporally-controlled capture of polyamide binding events, and (2) the biotin affinity handle is used to purify polyamide-DNA conjugates under semi-denaturing conditions to decrease DNA that is non-covalently bound. COSMIC is a general strategy that can be used to reveal the genome-wide binding events of polyamides and other genome-targeting chemotherapeutic agents.

Mapping polyamide-DNA interactions in human cells reveals a new design strategy for effective targeting of genomic sites.

Targeting the genome with sequence-specific synthetic molecules is a major goal at the interface of chemistry, biology, and personalized medicine. Pyrrole/imidazole-based polyamides can be rationally designed to target specific DNA sequences with exquisite precision in vitro; yet, the biological outcomes are often difficult to interpret using current models of binding energetics. To directly identify the binding sites of polyamides across the genome, we designed, synthesized, and tested polyamide derivatives that enabled covalent crosslinking and localization of polyamide-DNA interaction sites in live human cells. Bioinformatic analysis of the data reveals that clustered binding sites, spanning a broad range of affinities, best predict occupancy in cells. In contrast to the prevailing paradigm of targeting single high-affinity sites, our results point to a new design principle to deploy polyamides and perhaps other synthetic molecules to effectively target desired genomic sites in vivo.

Cooperativity in RNA-protein interactions: global analysis of RNA binding specificity.

The control and function of RNA are governed by the specificity of RNA binding proteins. Here, we describe a method for global unbiased analysis of RNA-protein interactions that uses in vitro selection, high-throughput sequencing, and sequence-specificity landscapes. The method yields affinities for a vast array of RNAs in a single experiment, including both low- and high-affinity sites. It is reproducible and accurate. Using this approach,we analyzed members of the PUF (Pumilio and FBF) family of eukaryotic mRNA regulators. Our data identify effects of a specific protein partner on PUF-RNA interactions, reveal subsets of target sites not previously detected, and demonstrate that designer PUF proteins can precisely alter specificity. The approach described here is, in principle, broadly applicable for analysis of any molecule that binds RNA, including proteins, nucleic acids, and small molecules.

Specificity landscapes of DNA binding molecules elucidate biological function.

Evaluating the specificity spectra of DNA binding molecules is a nontrivial challenge that hinders the ability to decipher gene regulatory networks or engineer molecules that act on genomes. Here we compare the DNA sequence specificities for different classes of proteins and engineered DNA binding molecules across the entire sequence space. These high-content data are visualized and interpreted using an interactive "specificity landscape" which simultaneously displays the affinity and specificity of a million-plus DNA sequences. Contrary to expectation, specificity landscapes reveal that synthetic DNA ligands match, and often surpass, the specificities of eukaryotic DNA binding proteins. The landscapes also identify differential specificity constraints imposed by diverse structural folds of natural and synthetic DNA binders. Importantly, the sequence context of a binding site significantly influences binding energetics, and utilizing the full contextual information permits greater accuracy in annotating regulatory elements within a given genome. Assigning such context-dependent binding values to every DNA sequence across the genome yields predictive genome-wide binding landscapes (genomescapes). A genomescape of a synthetic DNA binding molecule provided insight into its differential regulatory activity in cultured cells. The approach we describe will accelerate the creation of precision-tailored DNA therapeutics and uncover principles that govern sequence-specificity of DNA binding molecules.