Biology subjects

Shivalingam, A.

Publications and source records attributed to Shivalingam, A..

3 recordsLinked to original sources

bioRxiv2022

Transient DNA binding to gapped DNA substrates links DNA sequence to the single-molecule kinetics of protein-DNA interactions

Protein interactions with nucleic acids are central to all genetic processes and many biotechnological applications. While many sequence-dependent protein-DNA interactions have been studied in detail using single-molecule methods, there is no standard high-throughput way to link the complex single-molecule kinetics of protein-DNA interactions with the DNA sequence of a single molecule. Here we provide the missing link by introducing a single-molecule imaging method (Gap-Seq) that interrogates DNA sequences via transient binding of short fluorescent DNA to a single DNA molecule previously used to characterise a protein-DNA interaction. In Gap-Seq, we identify a base by the degree of binding of 6-9 nt-long DNAs to surface-immobilised DNA substrates featuring a short single-stranded gap. To facilitate detection, we also developed a fluorescence quenching strategy that allows single-molecule detection at up to 500 nM of unbound fluorescent DNA. We link single-base differences on single DNA molecules to the kinetics of protein-DNA interactions by studying the interaction of a transcription activator with its cognate site. Finally, we show that our assay can address mixed sequences by distinguishing between two different sequences immobilised on the same field of view, paving the way for interrogation of sequence libraries for both mechanistic work and biotechnological applications.

biophysics↗

bioRxiv2020

Targeted high-resolution chromosome conformation capture at genome-wide scale.

Chromosome conformation capture (3C) provides an adaptable tool for studying diverse biological questions. Current 3C methods provide either low-resolution interaction profiles across the entire genome, or high-resolution interaction profiles at up to several hundred loci. All 3C methods are affected to varying degrees by inefficiency, bias and noise. As such, generation of reproducible high-resolution interaction profiles has not been achieved at scale. To overcome this barrier, we systematically tested and improved upon current methods. We show that isolation of 3C libraries from intact nuclei, as well as shortening and titration of enrichment oligonucleotides used in high-resolution methods reduces noise and increases on-target sequencing. We combined these technical modifications into a new method Nuclear-Titrated (NuTi) Capture-C, which provides a >3-fold increase in informative sequencing content over current Capture-C protocols. Using NuTi Capture-C we target 8,061 promoters in triplicate, demonstrating that this method generates reproducible high-resolution genome-wide 3C interaction profiles at scale.

genomics↗

bioRxiv2019

Dissection of the 4D chromatin structure of the α-globin locus through in vivo erythroid differentiation with extreme spatial and temporal resolution

Mammalian gene expression patterns are controlled by regulatory elements, which interact within Topologically Associating Domains (TADs). The relationship between activation of regulatory elements, formation of structural chromatin interactions and gene expression during development is unclear. We developed Tiled-C, a low-input Chromosome Conformation Capture (3C) approach, to study chromatin architecture at high spatial and temporal resolution through in vivo mouse erythroid differentiation. Integrated analysis of matched chromatin accessibility and single-cell expression data shows that regulatory elements gradually become accessible within pre-existing TADs during early differentiation. This is followed by structural re-organization within the TAD and formation of specific contacts between enhancers and promoters. In contrast to previous reports, our high-resolution data show that these enhancer-promoter interactions are not established prior to gene expression, but formed gradually during differentiation, concomitant with progressive upregulation of gene activity. Together, these results provide new insight into the close, interdependent relationship between chromatin architecture and gene regulation during development.

genomics↗