Synthetic genome readers target clustered binding sites across diverse chromatin states

Abstract

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.

Specificity landscapes unmask submaximal binding site preferences of transcription factors

“…To elucidate similarities and differences in DPIs obtained by various experimental methods and sequence preferences of highly homologous TFs, we now report the development of DiSELs (Fig. 1B) (29). To perform DiSEL, we normalize paired DPI datasets and scale the dynamic range of one DPI against the other (Methods has details).…”

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

“…The lack of clarity on the parameters that govern genome-wide binding of polyamides greatly impedes the deployment of this class of molecules to regulate cell fate-defining and diseasecausing gene networks in vivo. 4 We report here the genome-wide binding profiles of two Py-Im polyamides 1 and 2, of identical architecture (8-ring hairpin) that differ at a single aromatic ring position in cellular nuclei using COSMIC-seq ('crosslinking of small molecules for isolation of chromatin with nextgeneration sequencing), Fig 1 [23,24]. COSMIC-seq employs a tripartite conjugate composed of the DNA-binding ligand attached to a biotin affinity handle and a psoralen photocrosslinker.…”

“…Genome-wide binding of these tripartite molecules is captured by photo-induced crosslinking followed by biotin-enabled enrichment and unbiased NGS sequencing of the conjugated genomic loci [23,24]. The ability to induce rapid crosslinking at the desired time point distinguishes COSMIC-seq from continuous and uncontrolled alkylation-dependent DNA conjugations that have been used to query genome-wide binding of polyamides [25].…”

“…COSMIC-seq also differs from Chem-seq approaches that use ligands for protein complexes that are associated with the genome [26]. Previously, COSMIC-seq was utilized to access genome-wide binding of two structurally distinct Py-Im polyamides (hairpin vs linear) that code for very different sequences [24]. An 8-ring hairpin Py-Im polyamide (TpPyPyIm-γ-PyImPyPyβ-Dp) binds 6 bp of DNA (5'-WTWCGW-3') [27], whereas a linear polyamide (ImPy-β-ImPyβ-Im-β-Dp) binds 9 bp of purine rich DNA (5'-AAGAAGAAG-3') [28][29][30][31].…”

“…Summation of sites (SOS) model was used to predict DNA binding of polyamides in the human genome, hg19 [23,24,43] . The SOS score was obtained by summing (or averaging) all k-mer in vitro binding intensities (enrichment) obtained using a sliding k-mer window across a genomic region.…”